# Patent

## Title
miRNA modulators of thermogenesis

## Abstract

Provided are novel methods and compositions for the modulation of thermogenesis. Such methods are particularly advantageous in that they allow for the reduction of body fat in a subject without the subject having to adjust their caloric intake through dieting, modify their physical activity or undergo bariatric surgery. Accordingly, the methods of the invention are particularly useful for treating or preventing obesity. Also provided are methods of screening for novel agents that modulate the activity of thermogenic regulators.

## Background
BACKGROUND OF THE INVENTION
Obesity has reached pandemic proportions, affecting all ages and socioeconomic groups. The World Health Organization estimated that in 2008, 1.5 billion adults aged 20 years and older were overweight and over 200 million men and 300 million women were obese. These figures are estimated to increase to 2.16 billion overweight and 1.12 billion obese individuals by 2030. Obesity is the source of lost earnings, restricted activity days, absenteeism, lower productivity at work (presenteeism), reduced quality of life, permanent disability, significant morbidity and mortality, and shortened lifespan. Indeed, the total annual economic cost of overweight and obesity in the United States and Canada caused by medical costs, excess mortality and disability was estimated to be about $300 billion in 2009. International studies on the economic costs of obesity have shown that they account for between 2% and 10% of total health care costs.
Obesity is the result of a chronic imbalance between energy intake and expenditure. This leads to storage of excess energy into adipocytes, which typically exhibit both hypertrophy (increase in cell size) and hyperplasia (increase in cell number or adipogenesis). The recent worsening of obesity is due to the combination of excessive consumption of energy-dense foods high in saturated fats and sugars, and reduced physical activity.
The current symptomatic medical treatments of obesity fail to achieve their long-term therapeutic goals, largely due to limited drug efficacy and patients' poor adherence with lifestyle changes and therapies. Several obesity drugs have been removed from the market for safety reasons and small molecules currently in development are struggling to gain regulatory approval because of their modest short-term efficacy and unknown safety profile. Presently, only restrictive and malabsorptive bariatric surgery can achieve significant long-term reduction of weight excess with some favorable cardiovascular benefits.
Accordingly, there is a need in the art for novel treatments for obesity.
SUMMARY OF THE INVENTION
Obesity is the consequence of a chronic imbalance of energy intake over expenditure, leading to the storage of excess energy inside white adipocytes. This disclosure features a novel treatment for obesity targeting peripheral adipocytes, including energy-storing lipid-filled white adipocytes (WAT), and energy-expending mitochondria-rich brown adipocytes (BAT). In addition, the disclosure provides methods for the modulation of thermogenesis (the process of heat production in organisms) using microRNA (miRNAs) agents. The methods described herein generally involve the direct and/or indirect modulation of at least one thermogenic regulator (e.g., a mitochondrial uncoupler, such as Uncoupling Protein 1 (UCP1 also known as Thermogenin) or Uncoupling Protein 2 (UCP2)) in a cell, tissue and/or subject using an isolated miRNA agent. UCPs uncouple oxidative phosphorylation from ATP synthesis. In certain instances, this uncoupling results in energy dissipated as heat. Such methods are particularly advantageous in that they allow for the reduction of body fat in a subject without the subject having to adjust their caloric intake through dieting, modify their physical activity or undergo bariatric surgery. Accordingly, the methods of the invention are particularly useful for treating or preventing obesity.
The invention also provides novel miRNA agent compositions (e.g., miRNA, agomirs, and antagomirs) that can modulate the activity of thermogenic regulators. Yet further, the invention provides methods of screening for novel miRNA agents that modulate the activity of thermogenic regulators. Further still, the invention provides novel agent compositions (e.g. aptamer-miRNA complexes or “aptamirs”) that provide cell/tissue-specific delivery of the miRNA agents.
Accordingly, in one aspect, the invention provides a method of modulating respiratory chain uncoupling in a cell, the method comprising contacting the cell with an isolated miRNA agent that modulates the expression level and/or activity of at least one mitochondrial uncoupler. In some embodiments, the method further comprises the step of selecting a subject in need of modulating respiratory chain uncoupling (e.g., an obese patient). In one embodiment, the miRNA agent increases the expression level and/or activity of the at least one mitochondrial uncoupler. In certain embodiments, the mitochondrial uncoupler is UCP1 or UCP2. In some embodiments, the method increases respiratory chain uncoupling in a cell in vivo. In other embodiments, the method increases respiratory chain uncoupling in a cell ex vivo. In certain embodiments, the method further comprises determining the level of expression (mRNA or protein) or activity of the mitochondrial uncoupler. In certain embodiments, the cell is a pre-adipocyte, adipocyte, adipose tissue derived mesenchymal stem cell, hepatocyte, myocyte, or a precursor thereof. Optionally, adipocytes can be white fat or brown fat adipocytes.
In another aspect, the invention provides a method of modulating thermogenesis in a tissue, the method comprising contacting the tissue with an isolated miRNA agent that modulates the expression level and/or activity of at least one mitochondrial uncoupler. In some embodiments, the method further comprises the step of selecting a subject in need of modulating thermogenesis (e.g., an obese patient). In one embodiment, the miRNA agent increases the expression level and/or activity of the at least one mitochondrial uncoupler. In certain embodiments, the mitochondrial uncoupler is UCP1 or UCP2. In certain embodiments, the method involves increasing thermogenesis. In certain embodiments, the method further comprises determining the level of expression (mRNA or protein) or activity of the mitochondrial uncoupler. In certain embodiments, the tissue is brown fat, white fat, subcutaneous adipose tissue, liver or muscle. In certain embodiments, the tissue is contacted with the miRNA agent ex vivo.
In another aspect, the invention provides a method of treating obesity in human subject in need of treatment thereof, the method generally comprising administering to the human subject an effective amount of a miRNA agent that modulates activity of at least one mitochondrial uncoupler. In certain embodiments, the human subject selected for treatment has a genetic or epigenetic predisposition to obesity. In certain embodiments, the mitochondrial uncoupler is UCP1 or UCP2.
In certain embodiments of all of the above aspects, the miRNA agent is an isolated miRNA selected from the group consisting of hsa-miR-1-1, hsa-miR-1-2, miR-19a-b, hsa-miR-105, hsa-miR-1283, hsa-mir-129, hsa-miR-133a-1, hsa-miR-133a-2, hsa-miR-143, hsa-mir-143-5p, hsa-mir-147, hsa-mir-149, hsa-mir-199a, hsa-mir-199b, hsa-mir-200c, hsa-mir-204, hsa-mir-205, hsa-miR-206, hsa-mir-21, hsa-mir-211, hsa-mir-218, hsa-mir-218-1, hsa-mir-218-2, hsa-mir-219-2, hsa-mir-219-2-3p, hsa-mir-22, hsa-mir-22-3p, hsa-mir-22-5p, hsa-mir-24-2, hsa-miR-30a-e, hsa-miR-3177-5p, hsa-mir-325, hsa-mir-331, hsa-mir-331-5p, hsa-miR-3613-3p, hsa-mir-362, hsa-mir-362-5p, hsa-miR-3658, hsa-mir-367, hsa-mir-371, hsa-mir-371-5p, hsa-mir-377, hsa-mir-378, hsa-mir-378a-5p, hsa-mir-382, hsa-mir-383, hsa-mir-422a, hsa-mir-425, hsa-miR-455-3p, hsa-miR-455-5p, hsa-miR-491, hsa-mir-508, hsa-mir-508-5p, hsa-mir-512-1, hsa-mir-512-2, hsa-miR-515-3p, hsa-mir-519e, hsa-miR-520a, hsa-mir-543, hsa-mir-545, hsa-mir-549, hsa-mir-556, and hsa-miR-568, hsa-mir-620, hsa-mir-643, hsa-mir-654-3p, hsa-miR-7a-g, hsa-mir-765, hsa-mir-871, hsa-mir-888, hsa-mir-888-3p, hsa-mir-92b, hsa-mir-93, hsa-mir-96, and hsa-mir-99a. In certain embodiments of all of the above aspects, the miRNA agent is an isolated miRNA that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of a miRNA listed above. In certain embodiments of all of the above aspects, the miRNA agent is a seed sequence of a miRNA listed above.
In certain embodiments of all of the above aspects, the miRNA agent is an isolated miRNA selected from the group consisting of the 536 miRNAs set forth in Table A. In certain embodiments of all of the above aspects, the miRNA agent is an isolated miRNA that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of a miRNA listed in Table A. In certain embodiments of all of the above aspects, the miRNA agent is a seed sequence of a miRNA listed in Table A.







TABLE A





Adipocyte miRNAs listed in ascending order (miRBase 19 nomenclature)


















hsa-let-7a-3p
hsa-miR-185-5p
hsa-miR-329
hsa-miR-509-3p


hsa-let-7a-5p
hsa-miR-186-3p
hsa-miR-330-3p
hsa-miR-511


hsa-let-7b-3p
hsa-miR-186-5p
hsa-miR-330-5p
hsa-miR-513a-3p


hsa-let-7b-5p
hsa-miR-187-3p
hsa-miR-331-3p
hsa-miR-513a-5p


hsa-let-7c
hsa-miR-188-5p
hsa-miR-331-5p
hsa-miR-513b


hsa-let-7d-3p
hsa-miR-18a-3p
hsa-miR-335-3p
hsa-miR-514a-3p


hsa-let-7d-5p
hsa-miR-18a-5p
hsa-miR-335-5p
hsa-miR-515-3p


hsa-let-7e-5p
hsa-miR-18b-5p
hsa-miR-337-3p
hsa-miR-516b-





3p


hsa-let-7f-1-3p
hsa-miR-1909-3p
hsa-miR-337-5p
hsa-miR-516b-





5p


hsa-let-7f-5p
hsa-miR-190a
hsa-miR-338-3p
hsa-miR-518b


hsa-let-7g-3p
hsa-miR-190b
hsa-miR-338-5p
hsa-miR-518e-3p


hsa-let-7g-5p
hsa-miR-191-3p
hsa-miR-339-3p
hsa-miR-518e-5p


hsa-let-7i-3p
hsa-miR-191-5p
hsa-miR-339-5p
hsa-miR-518f-3p


hsa-let-7i-5p
hsa-miR-192-5p
hsa-miR-33a-5p
hsa-miR-519a-5p


hsa-miR-1
hsa-miR-193a-3p
hsa-miR-33b-5p
hsa-miR-519b-





5p


hsa-miR-100-5p
hsa-miR-193a-5p
hsa-miR-340-3p
hsa-miR-519c-3p


hsa-miR-101-3p
hsa-miR-193b-3p
hsa-miR-340-5p
hsa-miR-519c-5p


hsa-miR-101-5p
hsa-miR-193b-5p
hsa-miR-342-3p
hsa-miR-519d


hsa-miR-103a-
hsa-miR-194-5p
hsa-miR-342-5p
hsa-miR-520c-3p


2-5p





hsa-miR-103a-3p
hsa-miR-195-3p
hsa-miR-345-5p
hsa-miR-520e


hsa-miR-103b
hsa-miR-195-5p
hsa-miR-346
hsa-miR-520f


hsa-miR-105-5p
hsa-miR-196a-5p
hsa-miR-34a-5p
hsa-miR-520g


hsa-miR-106a-5p
hsa-miR-196b-5p
hsa-miR-34b-3p
hsa-miR-520h


hsa-miR-106b-3p
hsa-miR-197-3p
hsa-miR-34b-5p
hsa-miR-521


hsa-miR-106b-5p
hsa-miR-198
hsa-miR-34c-5p
hsa-miR-522-5p


hsa-miR-107
hsa-miR-199a-3p
hsa-miR-3545-5p
hsa-miR-523-5p


hsa-miR-10a-3p
hsa-miR-199a-5p
hsa-miR-3591-3p
hsa-miR-525-3p


hsa-miR-10a-5p
hsa-miR-199b-3p
hsa-miR-361-3p
hsa-miR-532-3p


hsa-miR-10b-3p
hsa-miR-199b-5p
hsa-miR-361-5p
hsa-miR-532-5p


hsa-miR-10b-5p
hsa-miR-19a-3p
hsa-miR-3613-5p
hsa-miR-539-5p


hsa-miR-1179
hsa-miR-19b-3p
hsa-miR-3615
hsa-miR-542-3p


hsa-miR-1185-5p
hsa-miR-200a-3p
hsa-miR-362-3p
hsa-miR-542-5p


hsa-miR-1208
hsa-miR-200a-5p
hsa-miR-362-5p
hsa-miR-545-3p


hsa-miR-122-5p
hsa-miR-200b-3p
hsa-miR-363-3p
hsa-miR-545-5p


hsa-miR-1227-3p
hsa-miR-200c-3p
hsa-miR-363-5p
hsa-miR-548d-





3p


hsa-miR-1228-5p
hsa-miR-202-3p
hsa-mir-365a-3p
hsa-miR-548e


hsa-miR-1229-3p
hsa-miR-203a
hsa-mir-3653
hsa-miR-548i


hsa-miR-124-3p
hsa-miR-204-5p
hsa-miR-3656
hsa-miR-548m


hsa-miR-125a-3p
hsa-miR-205-5p
hsa-miR-365a-3p
hsa-miR-550a-5p


hsa-miR-125a-5p
hsa-miR-206
hsa-miR-365a-5p
hsa-miR-551b-





3p


hsa-miR-125b-
hsa-miR-20a-3p
hsa-miR-367-3p
hsa-miR-552


1-3p





hsa-miR-125b-
hsa-miR-20a-5p
hsa-mir-3676-3p
hsa-miR-553


2-3p





hsa-miR-125b-5p
hsa-miR-20b-5p
hsa-miR-369-3p
hsa-miR-554


hsa-miR-126-3p
hsa-miR-21-3p
hsa-miR-369-5p
hsa-miR-557


hsa-miR-126-5p
hsa-miR-21-5p
hsa-miR-370
hsa-miR-563


hsa-miR-1260a
hsa-miR-210
hsa-miR-371a-3p
hsa-miR-564


hsa-miR-1260b
hsa-miR-211-5p
hsa-miR-373-3p
hsa-miR-567


hsa-miR-1268a
hsa-miR-2110
hsa-miR-373-5p
hsa-miR-569


hsa-miR-127-3p
hsa-miR-212-3p
hsa-miR-374a-3p
hsa-miR-570-3p


hsa-miR-127-5p
hsa-miR-214-3p
hsa-miR-374a-5p
hsa-miR-572


hsa-miR-1271-5p
hsa-miR-214-5p
hsa-miR-374b-3p
hsa-miR-574-3p


hsa-miR-1273a
hsa-miR-215
hsa-miR-374b-5p
hsa-miR-574-5p


hsa-miR-1277-3p
hsa-miR-216a-5p
hsa-miR-375
hsa-miR-575


hsa-miR-128
hsa-miR-217
hsa-mir-376a-2-
hsa-miR-576-




5p
3p


hsa-miR-128-2
hsa-miR-218-5p
hsa-miR-376a-3p
hsa-miR-576-5p


hsa-miR-1285-3p
hsa-miR-219-1-3p
hsa-miR-376a-5p
hsa-miR-582-3p


hsa-miR-1287
hsa-miR-219-5p
hsa-miR-376b-3p
hsa-miR-582-5p


hsa-miR-1288
hsa-miR-22-3p
hsa-miR-376c-3p
hsa-miR-583


hsa-miR-129-5p
hsa-miR-22-5p
hsa-miR-377-3p
hsa-miR-584-5p


hsa-miR-1290
hsa-miR-221-3p
hsa-miR-378a-3p
hsa-miR-585


hsa-miR-1292-5p
hsa-miR-221-5p
hsa-miR-378a-5p
hsa-miR-586


hsa-miR-1301
hsa-miR-222-3p
hsa-miR-378c
hsa-miR-589-5p


hsa-miR-1305
hsa-miR-222-5p
hsa-miR-378d
hsa-miR-590-3p


hsa-mir-1307-3p
hsa-miR-223-3p
hsa-miR-379-5p
hsa-miR-590-5p


hsa-miR-130a-3p
hsa-miR-223-5p
hsa-miR-380-3p
hsa-miR-595


hsa-miR-130b-3p
hsa-miR-224-3p
hsa-miR-381-3p
hsa-miR-598


hsa-miR-130b-5p
hsa-miR-224-5p
hsa-miR-382-5p
hsa-miR-601


hsa-miR-132-3p
hsa-miR-2355-3p
hsa-miR-383
hsa-miR-602


hsa-miR-132-5p
hsa-miR-23a-3p
hsa-miR-384
hsa-miR-603


hsa-miR-1323
hsa-miR-23b-3p
hsa-miR-3912
hsa-miR-605


hsa-miR-133a
hsa-miR-23b-5p
hsa-miR-3928
hsa-miR-606


hsa-miR-133b
hsa-miR-24-1-5p
hsa-miR-409-3p
hsa-miR-609


hsa-miR-134
hsa-miR-24-2-5p
hsa-miR-409-5p
hsa-miR-611


hsa-miR-135a-5p
hsa-miR-24-3p
hsa-miR-410
hsa-miR-615-3p


hsa-miR-135b-5p
hsa-miR-25-3p
hsa-miR-411-5p
hsa-miR-619


hsa-miR-136-3p
hsa-miR-26a-2-3p
hsa-miR-421
hsa-miR-625-5p


hsa-miR-136-5p
hsa-miR-26a-5p
hsa-miR-422a
hsa-miR-627


hsa-miR-137
hsa-miR-26b-3p
hsa-miR-422b
hsa-miR-628-3p


hsa-miR-138-
hsa-miR-26b-5p
hsa-miR-423-3p
hsa-miR-628-5p


1-3p





hsa-miR-138-5p
hsa-miR-27a-3p
hsa-miR-423-5p
hsa-miR-629-3p


hsa-miR-139-3p
hsa-miR-27a-5p
hsa-miR-424-3p
hsa-miR-629-5p


hsa-miR-139-5p
hsa-miR-27b-3p
hsa-miR-424-5p
hsa-miR-630


hsa-miR-140-3p
hsa-miR-27b-5p
hsa-miR-425-3p
hsa-miR-636


hsa-miR-140-5p
hsa-miR-28-3p
hsa-miR-425-5p
hsa-miR-638


hsa-miR-141-3p
hsa-miR-28-5p
hsa-miR-429
hsa-miR-639


hsa-miR-142-3p
hsa-miR-296-5p
hsa-miR-431-5p
hsa-miR-641


hsa-miR-142-5p
hsa-miR-297
hsa-miR-432-5p
hsa-miR-642a-3p


hsa-miR-143-3p
hsa-miR-298
hsa-miR-433
hsa-miR-642a-5p


hsa-miR-143-5p
hsa-miR-299-3p
hsa-miR-4421
hsa-miR-646


hsa-miR-144-3p
hsa-miR-299-5p
hsa-miR-449a
hsa-miR-649


hsa-miR-144-5p
hsa-miR-29a-3p
hsa-miR-450a-5p
hsa-miR-651


hsa-miR-145-3p
hsa-miR-29a-5p
hsa-miR-450b-3p
hsa-miR-652-3p


hsa-miR-145-5p
hsa-miR-29b-1-5p
hsa-miR-450b-5p
hsa-miR-653


hsa-miR-1468
hsa-miR-29b-2-5p
hsa-miR-4510
hsa-miR-654-3p


hsa-miR-146a-5p
hsa-miR-29b-3p
hsa-miR-4516
hsa-miR-659-3p


hsa-miR-146b-3p
hsa-miR-29c-3p
hsa-miR-451a
hsa-miR-660-5p


hsa-miR-146b-5p
hsa-miR-29c-5p
hsa-miR-452-3p
hsa-miR-663a


hsa-miR-147a
hsa-miR-301a-3p
hsa-miR-452-5p
hsa-miR-664a-3p


hsa-miR-148a-3p
hsa-miR-301b
hsa-miR-454-3p
hsa-miR-664a-5p


hsa-miR-148a-5p
hsa-miR-302a-5p
hsa-miR-454-5p
hsa-miR-668


hsa-miR-148b-3p
hsa-miR-302b-5p
hsa-miR-455-3p
hsa-miR-671-5p


hsa-miR-148b-5p
hsa-miR-302c-5p
hsa-miR-455-5p
hsa-miR-675-3p


hsa-miR-149-5p
hsa-miR-302d-3p
hsa-miR-4634
hsa-miR-675-5p


hsa-miR-150-3p
hsa-miR-3065-3p
hsa-miR-4732-5p
hsa-miR-7-2-3p


hsa-miR-150-5p
hsa-miR-3065-5p
hsa-miR-4792
hsa-miR-7-5p


hsa-miR-151a-3p
hsa-miR-3074-3p
hsa-miR-483-3p
hsa-miR-708-3p


hsa-miR-151a-5p
hsa-miR-3074-5p
hsa-miR-483-5p
hsa-miR-708-5p


hsa-miR-151b
hsa-miR-30a-3p
hsa-miR-484
hsa-miR-718


hsa-miR-152
hsa-miR-30a-5p
hsa-miR-485-5p
hsa-miR-744-5p


hsa-miR-153
hsa-miR-30b-3p
hsa-miR-486-3p
hsa-miR-765


hsa-miR-1539
hsa-miR-30b-5p
hsa-miR-486-5p
hsa-miR-769-5p


hsa-miR-154-3p
hsa-miR-30c-1-3p
hsa-miR-487b
hsa-miR-770-5p


hsa-miR-154-5p
hsa-miR-30c-2-3p
hsa-miR-488-3p
hsa-miR-874


hsa-miR-155-5p
hsa-miR-30c-5p
hsa-miR-489
hsa-miR-885-3p


hsa-miR-15a-3p
hsa-miR-30d-3p
hsa-miR-491-3p
hsa-miR-887


hsa-miR-15a-5p
hsa-miR-30d-5p
hsa-miR-491-5p
hsa-miR-889


hsa-miR-15b-3p
hsa-miR-30e-3p
hsa-miR-492
hsa-miR-890


hsa-miR-15b-5p
hsa-miR-30e-5p
hsa-miR-493-3p
hsa-miR-891a


hsa-miR-16-1-3p
hsa-miR-31-3p
hsa-miR-493-5p
hsa-miR-891b


hsa-miR-16-2-3p
hsa-miR-31-5p
hsa-miR-494
hsa-miR-9-5p


hsa-miR-16-5p
hsa-miR-3120-3p
hsa-miR-495-3p
hsa-miR-92a-3p


hsa-miR-17-3p
hsa-miR-3120-5p
hsa-miR-497-5p
hsa-miR-92b-3p


hsa-miR-17-5p
hsa-miR-3184-5p
hsa-miR-498
hsa-miR-93-3p


hsa-miR-181a-
hsa-miR-32-3p
hsa-miR-499a-5p
hsa-miR-93-5p


2-3p





hsa-miR-181a-3p
hsa-miR-32-5p
hsa-miR-500a-3p
hsa-miR-935


hsa-miR-181a-5p
hsa-miR-320a
hsa-miR-501-3p
hsa-miR-942


hsa-miR-181b-5p
hsa-miR-320b
hsa-miR-501-5p
hsa-miR-95


hsa-miR-181c-3p
hsa-miR-320c
hsa-miR-502-3p
hsa-miR-96-3p


hsa-miR-181c-5p
hsa-miR-323a-3p
hsa-miR-502-5p
hsa-miR-96-5p


hsa-miR-181d
hsa-miR-324-3p
hsa-miR-503-5p
hsa-miR-98-5p


hsa-miR-182-5p
hsa-miR-324-5p
hsa-miR-504
hsa-miR-99a-3p


hsa-miR-183-5p
hsa-miR-325
hsa-miR-505-3p
hsa-miR-99a-5p


hsa-miR-184
hsa-miR-326
hsa-miR-505-5p
hsa-miR-99b-3p


hsa-miR-185-3p
hsa-miR-328
hsa-miR-506-3p
hsa-miR-99b-5p









In certain embodiments of all of the above aspects, the miRNA agent is a miRNA selected from the group consisting of the isolated miRNAs set forth in Table 7. In certain embodiments of all of the above aspects, the miRNA agent is an isolated miRNA that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of a miRNA listed in Table 8. In certain embodiments of all of the above aspects, the miRNA agent is a seed sequence of a miRNA listed in Table 8.
In certain embodiments of all of the above aspects, the miRNA agent is an agomir or antagomir of a miRNA selected from the group consisting of the miRNAs set forth in Table A.
In certain embodiments of all of the above aspects, the miRNA agent is an agomir or antagomir of a miRNA selected from the group consisting of hsa-miR-1-1, hsa-miR-1-2, miR-19a-b, hsa-miR-105, hsa-miR-1283, hsa-mir-129, hsa-miR-133a-1, hsa-miR-133a-2, hsa-miR-143, hsa-mir-143-5p, hsa-mir-147, hsa-mir-149, hsa-mir-199a, hsa-mir-199b, hsa-mir-200c, hsa-mir-204, hsa-mir-205, hsa-miR-206, hsa-mir-21, hsa-mir-211, hsa-mir-218, hsa-mir-218-1, hsa-mir-218-2, hsa-mir-219-2, hsa-mir-219-2-3p, hsa-mir-22, hsa-mir-22-3p, hsa-mir-22-5p, hsa-mir-24-2, hsa-miR-30a-e, hsa-miR-3177-5p, hsa-mir-325, hsa-mir-331, hsa-mir-331-5p, hsa-miR-3613-3p, hsa-mir-362, hsa-mir-362-5p, hsa-miR-3658, hsa-mir-367, hsa-mir-371, hsa-mir-371-5p, hsa-mir-377, hsa-mir-378, hsa-mir-378a-5p, hsa-mir-382, hsa-mir-383, hsa-mir-422a, hsa-mir-425, hsa-miR-455-3p, hsa-miR-455-5p, hsa-miR-491, hsa-mir-508, hsa-mir-508-5p, hsa-mir-512-1, hsa-mir-512-2, hsa-miR-515-3p, hsa-mir-519e, hsa-miR-520a, hsa-mir-543, hsa-mir-545, hsa-mir-549, hsa-mir-556, and hsa-miR-568, hsa-mir-620, hsa-mir-643, hsa-mir-654-3p, hsa-miR-7a-g, hsa-mir-765, hsa-mir-871, hsa-mir-888, hsa-mir-888-3p, hsa-mir-92b, hsa-mir-93, hsa-mir-96, and hsa-mir-99a.
In certain embodiments of all of the above aspects, the miRNA agent is an agomir or antagomir of a miRNA selected from the group consisting of the miRNAs set forth in Table 7.
In certain embodiments of all of the above aspects, the miRNA agent is an antagomir of a miRNA selected from the group consisting of hsa-miR-19b-2-5p, hsa-miR-21-5p, hsa-miR-130b-5p, hsa-miR-211, hsa-miR-325, hsa-miR-382-3p/5p, hsa-miR-543, hsa-miR-515-3p, and hsa-miR-545.
In certain embodiments of all of the above aspects, the miRNA agent is an antagomir of a miRNA selected from the group consisting of hsa-miR-331-5p, hsa-miR-552, hsa-miR-620, and hsa-miR-1179.
In certain embodiments of all of the above aspects, the miRNA agent is linked to a targeting moiety (e.g., an aptamer). In one embodiment, the targeting moiety delivers the miRNA agent to a specific cell type or tissue.
In certain embodiments of all of the above aspects, the miRNA agent directly binds to the mRNA or promoter region of at least one mitochondrial uncoupler.
In certain embodiments of all of the above aspects, the miRNA agent directly binds to the 5′UTR or coding sequence of the mRNA of at least one mitochondrial uncoupler.
In certain embodiments of all of the above aspects, the miRNA agent directly binds to the 3′UTR of the mRNA of at least one mitochondrial uncoupler.
In certain embodiments of all of the above aspects, the miRNA agent modulates the activity of an activator or repressor of a mitochondrial uncoupling protein. In one embodiment, the miRNA agent directly binds to the mRNA or promoter region of the activator or repressor. In one embodiment, the miRNA agent directly binds to the 5′UTR or coding sequence of the mRNA of the activator or repressor. In one embodiment, the miRNA agent directly binds to the 3′UTR of the mRNA of the activator or repressor. In one embodiment, the activator or repressor is selected from the group listed in Table 1.
In certain embodiments of all of the above aspects, the mRNA or protein expression of the mitochondrial uncoupling protein is upregulated.
In certain embodiments of all of the above aspects, the mitochondrial uncoupling activity of the mitochondrial uncoupling protein is upregulated.
In another aspect, the invention provides a method of screening for a miRNA agent that modulates thermogenesis, the method generally comprising: providing an indicator cell; contacting the indicator cell with a test miRNA agent; and determining the cellular activity of at least one thermogenic regulator in the indicator cell in the presence and absence of the miRNA agent, wherein a change in the activity of the thermogenic regulator in the presence of the test miRNA agent identifies the test miRNA agent as a miRNA agent that modulates thermogenesis. The indicator cell can be a mammalian cell. In certain embodiments, the indicator cell is a human cell comprising at least a portion of a human genome.
In certain embodiments, the cell is a pre-adipocyte, adipocyte, adipose tissue derived mesenchymal stem cell, hepatocyte, myocyte, or a precursor thereof.
In certain embodiments, the cellular activity of the thermogenic regulator determined in the method is the mRNA expression level, protein expression level or mitochondrial uncoupling activity of the thermogenic regulator.
In certain embodiments, the test miRNA agent increases the activity of the thermogenic regulator compared to the level of activity of the thermogenic regulator in the absence of the test miRNA agent.
In certain embodiments, the thermogenic regulator is UCP1 or UCP2.
In another aspect, the invention provides an agomir or antagomir that modulates the activity of at least one thermogenic regulator in a cell.
In certain embodiments, the agomir or antagomir is an agomir or antagomir of a miRNA selected from the group consisting of the miRNAs set forth in Table 8.
In certain embodiments, the agomir or antagomir is an agomir or antagomir of a miRNA selected from the group consisting of the miRNAs set forth in Table A.
In certain embodiments, the agomir or antagomir is an agomir or antagomir of a miRNA selected from the group consisting of hsa-miR-1-1, hsa-miR-1-2, miR-19a-b, hsa-miR-105, hsa-miR-1283, hsa-mir-129, hsa-miR-133a-1, hsa-miR-133a-2, hsa-miR-143, hsa-mir-143-5p, hsa-mir-147, hsa-mir-149, hsa-mir-199a, hsa-mir-199b, hsa-mir-200c, hsa-mir-204, hsa-mir-205, hsa-miR-206, hsa-mir-21, hsa-mir-211, hsa-mir-218, hsa-mir-218-1, hsa-mir-218-2, hsa-mir-219-2, hsa-mir-219-2-3p, hsa-mir-22, hsa-mir-22-3p, hsa-mir-22-5p, hsa-mir-24-2, hsa-miR-30a-e, hsa-miR-3177-5p, hsa-mir-325, hsa-mir-331, hsa-mir-331-5p, hsa-miR-3613-3p, hsa-mir-362, hsa-mir-362-5p, hsa-miR-3658, hsa-mir-367, hsa-mir-371, hsa-mir-371-5p, hsa-mir-377, hsa-mir-378, hsa-mir-378a-5p, hsa-mir-382, hsa-mir-383, hsa-mir-422a, hsa-mir-425, hsa-miR-455-3p, hsa-miR-455-5p, hsa-miR-491, hsa-mir-508, hsa-mir-508-5p, hsa-mir-512-1, hsa-mir-512-2, hsa-miR-515-3p, hsa-mir-519e, hsa-miR-520a, hsa-mir-543, hsa-mir-545, hsa-mir-549, hsa-mir-556, and hsa-miR-568, hsa-mir-620, hsa-mir-643, hsa-mir-654-3p, hsa-miR-7a-g, hsa-mir-765, hsa-mir-871, hsa-mir-888, hsa-mir-888-3p, hsa-mir-92b, hsa-mir-93, hsa-mir-96, and hsa-mir-99a.
In certain embodiments, the agomir or antagomir is an antagomir of a miRNA selected from the group consisting of hsa-miR-19b-2-5p, ha-miR-21-5p, hsa-miR-130b-5p, hsa-miR-211, hsa-miR-325, hsa-miR-382-3p/5p, hsa-miR-543, hsa-miR-515-3p, and hsa-miR-545.
In certain embodiments, the agomir or antagomir is an antagomir of a miRNA selected from the group consisting of hsa-miR-331-5p, hsa-miR-552, hsa-miR-620, and hsa-miR-1179.
In certain embodiments, the agomir or antagomir is linked to a targeting moiety.
In certain embodiments, the targeting moiety is an aptamer.
In certain embodiments, the targeting moiety delivers the agomir or antagomir to a specific cell type or tissue.
In certain embodiments, the agomir or antagomir directly binds to the mRNA or promoter region of at least one mitochondrial uncoupler.
In certain embodiments, the agomir or antagomir directly binds to the 5′UTR or coding sequence of the mRNA of at least one mitochondrial uncoupler.
In certain embodiments, the agomir or antagomir directly binds to the 3′UTR of the mRNA of at least one mitochondrial uncoupler.
In certain embodiments, the agomir or antagomir modulates the activity of an activator or repressor of a mitochondrial uncoupling protein.
In certain embodiments, the activator or repressor is selected from the group listed in Table 1.
In certain embodiments, the agomir or antagomir directly binds to the mRNA or promoter region of the activator or repressor.
In certain embodiments, the agomir or antagomir directly binds to the 5′UTR or coding sequence of the mRNA of the activator or repressor. In other embodiments, the agomir or antagomir directly binds to the 3′UTR of the mRNA of the activator or repressor.
The disclosure also provides a pharmaceutical composition comprising two or more miRNAs selected from hsa-let-7a agomir, hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-1 antagomir, hsa-miR-19b agomir, hsa-miR-19b antagomir, hsa-miR-30b agomir and hsa-miR-30b antagomir. In certain embodiments the pharmaceutical composition also includes a pharmaceutically acceptable excipient. In certain embodiments, the two or more miRNAs are expressed from a recombinant vector. The recombinant vector can be selected from DNA plasmids, viral vectors and DNA minicircles.
The disclosure also provides a method of inducing pre-adipocytes to differentiate initially into white adipocytes and subsequently into brown adipocytes comprising administering to a population of pre-adipocytes one or more miRNAs selected from hsa-let-7a agomir, hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-1 antagomir, hsa-miR-19b agomir, hsa-miR-19b antagomir, hsa-miR-30b agomir and hsa-miR-30b antagomir. The one or more miRNAs can also be selected from hsa-let-7a agomir, hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-1 antagomir, hsa-miR-19b agomir, hsa-miR-19b antagomir, hsa-miR-30b agomir and hsa-miR-30b antagomir. In certain embodiments, the induction of pre-adipocytes to differentiate into adipocytes is greater than the differentiation of pre-adipocytes to adipocytes when pre-adipocytes are exposed to 100 mM rosiglitazone for two days followed by maintenance medium. In certain embodiments, the adipocytes are brown adipocytes. In other embodiments, the adipocytes are white adipocytes. Additional criteria for differentiation can be found in the Examples, below.
The disclosure also provides a method for decreasing the lipid content of adipocytes comprising administering to a population of adipocytes one or more miRNAs selected from the group consisting of hsa-let-7a agomir, hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-1 antagomir, hsa-miR-19b agomir, hsa-miR-19b antagomir, hsa-miR-30b agomir and hsa-miR-30b antagomir. In certain embodiments, the lipid content of the adipocytes is less than the lipid content of adipocytes exposed to 100 nM rosiglitazone for two days followed by maintenance medium or less than the fat content of adipocytes exposed to 100 nM rosiglitazone for the duration of culture. The duration of culture can be 8-16, 10-14 or 14 days. The duration of culture can also be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days. Additional criteria for lipid content of adipocytes can be found in the Examples, below.
The disclosure also provides a method for increasing insulin sensitivity in a subject in need thereof comprising administering the subject one or more miRNAs selected from the group consisting of hsa-let-7a agomir, hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-1 antagomir, hsa-miR-19b agomir, hsa-miR-19b antagomir, hsa-miR-30b agomir and hsa-miR-30b antagomir.
In certain embodiments, the subject is a mammal.
The disclosure also provides a method of increasing expression or activity of one or more uncoupling proteins in a cell comprising administering to the cell one or more, two or more, or three or more miRNAs selected from the group consisting of hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-19b agomir and hsa-miR-30b agomir. In certain embodiments, the cell is selected from the group consisting of a brown adipocyte, a white adipocyte, a subcutaneous adipocyte, a liver cell or a muscle cell. In other embodiments, the one or more uncoupling proteins include UCP1 or UCP2. In certain embodiments, the method is an ex vivo method. In other embodiments, the method is an in vivo method. In certain embodiments, the method involves selecting a subject (e.g., a human) in need of increasing the level of expression or activity of one or more uncoupling proteins (e.g., UCP1, UCP2). In some embodiments, the subject has, or is at risk of developing, obesity. In certain embodiments, the subject has, or is at risk of developing, diabetes. In certain embodiments, the method further comprises determining the expression level (mRNA or protein) or activity of the one or more uncoupling proteins.
The disclosure also provides a method of causing fat loss in a subject in need thereof comprising administering the subject one or more miRNAs selected from the group consisting of hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-19b agomir and hsa-miR-30b agomir. In certain embodiments, the subject is a mammal. In other embodiments, the mammal is a human.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B are schematic representations of the interactions of 83 thermogenic regulators determined using the STRING 9.0 database.
FIG. 2A is a schematic representation of the interaction of 83 thermogenic regulators determined using the Ingenuity Pathway Analysis Software program.
FIG. 2B is a schematic representation of the interaction of 83 thermogenic regulators determined using the Reactome Functional Interaction Network program.
FIG. 3 is a schematic representation of the overlap between the visual inspection and alignment of nucleotide sequences set forth herein, and the results from multiple miRNA prediction programs predicting miRNA binding sites in the 5′UTR, promoter region, coding sequence and 3′UTR of the human UCP1 gene.
FIG. 4 is a schematic representation of the overlap of results from multiple miRNA prediction programs predicting miRNA binding sites in the 5′UTR, promoter region, coding sequence and 3′UTR of the genes of 83 thermogenic regulators.
FIG. 5 is a schematic representation of oxidative phosphorylation in mitochondria, illustrating the uncoupling of oxidative phosphorylation from ATP synthesis by UCP1 to generate heat.
FIG. 6 depicts the transcriptional control of UCP1 by other exemplary thermogenic regulators.
FIG. 7 depicts exemplary positive (A) and negative (B) transcriptional regulators of the UCP1 gene.
FIG. 8A depicts the location of various regulatory elements in reference to the transcription start site in the 15,910 base pair (bp) human UCP1 gene sequence (NCBI Reference Sequence: gi|237858805|ref|NG—012139.1| Homo sapiens uncoupling protein 1 (mitochondrial, proton carrier) (UCP1), RefSeqGene on chromosome 4).
FIG. 8B depicts the location of various regulatory elements in reference to the transcription start site in the 15,174 bp of the human UCP2 gene (ENSG00000175567), including 5,000 bp 5′UTR and 2,000 bp 3′UTR on chromosome 11.
FIG. 9 is a bar graph showing relative fluorescence in unlabeled cells or cells transfected with a Dy547 labeled non-targeting miRIDIAN mimic and hairpin inhibitor.
FIG. 10A is a bar graph showing the reduction of GAPDH expression in cells transfected with siRNA control and a GAPDH siRNA 4 days after transfection.
FIG. 10B is a bar graph showing the reduction of GAPDH expression in cells transfected with siRNA control and a GAPDH siRNA 12 days after transfection.
FIG. 11A is a light micrograph of preadipocytes stained with Oil Red O cultured for 2 weeks in maintenance medium without rosiglitazone.
FIG. 11B is a light micrograph of preadipocytes stained with Oil Red O cultured in the presence of insulin, triiodothyronine, dexamethasone, isobutyl-methylxanthine and rosiglitazone for two days followed by maintenance medium for 12 days.
FIG. 11C is a light micrograph of preadipocytes stained with Oil Red O cultured in the presence of insulin, triiodothyronine, dexamethasone, isobutyl-methylxanthine and rosiglitazone throughout the experiment.
FIG. 11D is a light micrograph of preadipocytes stained with Oil Red O cultured in the presence of hsa-miR-30b mimic.
FIG. 11E is a light micrograph of preadipocytes stained with Oil Red O cultured in the presence of non targeting miRNA mimic.
FIG. 11F is a light micrograph of preadipocytes stained with Oil Red O cultured in the presence of non targeting miRNA inhibitor.
FIG. 12A is a bar graph showing mRNA expression of thermogenesis targets in the presence of rosiglitazone.
FIG. 12B is a bar graph showing mRNA expression of thermogenesis targets in the presence of hsa-let-7a inhibitor.
FIG. 12C is a bar graph showing mRNA expression of thermogenesis targets in the presence of hsa-miR-1 mimic.
FIG. 12D is a bar graph showing mRNA expression of thermogenesis targets in the presence of hsa-miR-19b mimic.
FIG. 12E is a bar graph showing mRNA expression of thermogenesis targets in the presence of and hsa-miR-30b mimic.
FIG. 12F is a bar graph showing mRNA expression of thermogenesis targets in untreated preadipocytes.
FIG. 13 is a bar graph showing relative fluorescence in unlabeled cells and cells transfected with a Dy547 labeled non-targeting miRIDIAN mimic or hairpin inhibitor.
FIG. 14A is a bar graph showing the reduction of GAPDH expression in cells transfected with siRNA control and a GAPDH siRNA 4 days after transfection.
FIG. 14B is a bar graph showing the reduction of GAPDH expression in cells transfected with siRNA control and a GAPDH siRNA 12 days after transfection.
FIG. 15 is a bar graph showing the amount of lipids in mature adipocytes using Nile Red Dye exposed to various miRNAs.
FIG. 16 is an M-A plot showing the mean gene expression on the x-axis and the difference between pairs in logarithmic scale on the y-axis.
FIG. 17 is a schematic showing a Venn Diagram showing that the numbers of genes significantly upregulated in the presence of the miRNA analogs hsa-let-7a inhibitor, hsa-miR-1 mimic, hsa-miR-19b mimic and hsa-miR-30b mimic were respectively 305, 247, 255 and 267. A set of 127 genes was commonly upregulated by the listed miRNA analogs.
FIG. 18 is a schematic showing a Venn diagram showing that the numbers of genes significantly downpregulated in the presence of the miRNA analogs hsa-let-7a inhibitor, hsa-miR-1 mimic, hsa-miR-19b mimic and hsa-miR-30b mimic were respectively 143, 177, 115 and 165. A set of 60 genes that was commonly downregulated by the listed miRNA analogs.
FIG. 19 is a bar graph showing the amounts of RNA extracted from mature adipocytes exposed to various transfecting agents.
FIG. 20 is a bar graph showing reduction of GAPDH expression in mature adipocytes transfected with a GAPDH-specific miRNA mimic using various transfecting agents.



DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application, including definitions, will control.
As used herein, the term “miRNA agent” refers to an oligonucleotide or oligonucleotide mimetic that directly or indirectly modulates the activity of a thermogenic regulator (e.g., a mitochondrial uncoupler or an activator or repressor thereof). miRNA agents can act on a target gene or on a target miRNA.
As used herein, the term “miRNA” refers to a single-stranded RNA molecule (or a synthetic derivative thereof), which is capable of binding to a target gene (either the mRNA or the DNA) and regulating expression of that gene. In certain embodiments, the miRNA is naturally expressed in an organism.
As used herein, the term “seed sequence” refers to a 6-8 nucleotide (nt) long substring within the first 8 nt at the 5′-end of the miRNA (i.e., seed sequence) that is an important determinant of target specificity.
As used herein, the term “agomir” refers to a synthetic oligonucleotide or oligonucleotide mimetic that functionally mimics a miRNA. An agomir can be an oligonucleotide with the same or similar nucleic acid sequence to a miRNA or a portion of a miRNA. In certain embodiments, the agomir has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide differences from the miRNA that it mimics. Further, agomirs can have the same length, a longer length or a shorter length than the miRNA that it mimics. In certain embodiments, the agomir has the same sequence as 6-8 nucleotides at the 5′ end of the miRNA it mimics. In other embodiments, an agomir can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In other embodiments, an agomir can be 5-10, 6-8, 10-20, 10-15 or 5-500 nucleotides in length. In certain embodiments, agomirs include any of the sequences shown in Table A. These chemically modified synthetic RNA duplexes include a guide strand that is identical or substantially identical to the miRNA of interest to allow efficient loading into the miRISC complex, whereas the passenger strand is chemically modified to prevent its loading to the Argonaute protein in the miRISC complex (Thorsen S B et al., Cancer J., 18(3):275-284 (2012); Broderick J A et al., Gene Ther., 18(12):1104-1110 (2011)).
As used herein, the term “antagomir” refers to a synthetic oligonucleotide or oligonucleotide mimetic having complementarity to a specific microRNA, and which inhibits the activity of that miRNA. In certain embodiments, the antagomir has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide differences from the miRNA that it inhibits. Further, antagomirs can have the same length, a longer length or a shorter length than the miRNA that it inhibits. In certain embodiments, the antagomir hybridizes to 6-8 nucleotides at the 5′ end of the miRNA it inhibits. In other embodiments, an antagomir can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In other embodiments, an antagomir can be 5-10, 6-8, 10-20, 10-15 or 5-500 nucleotides in length. In certain embodiments, antagomirs include nucleotides that are complementary to any of the sequences shown in Table A. The antagomirs are synthetic reverse complements that tightly bind to and inactivate a specific miRNA. Various chemical modifications are used to improve nuclease resistance and binding affinity. The most commonly used modifications to increase potency include various 2′ sugar modifications, such as 2′-O-Me, 2′-O-methoxyethyl (2′-MOE), or 2′-fluoro(2′-F). The nucleic acid structure of the miRNA can also be modified into a locked nucleic acid (LNA) with a methylene bridge between the 2′ oxygen and the 4′ carbon to lock the ribose in the 3′-endo (North) conformation in the A-type conformation of nucleic acids (Lennox K A et al. Gene Ther. December 2011; 18(12):1111-1120; Bader A G et al. Gene Ther. December 2011; 18(12):1121-1126). This modification significantly increases both target specificity and hybridization properties of the molecules.
As used herein, the term “aptamir” refers to the combination of an aptamer (oligonucleic acid or peptide molecule that bind to a specific target molecule) and an agomir or antagomir as defined above, which allows cell or tissue-specific delivery of the miRNA agents.
As used herein, the term “interfering RNA” refers to any double stranded or single stranded RNA sequence capable of inhibiting or down-regulating gene expression by mediating RNA interference. Interfering RNAs include, but are not limited to, small interfering RNA (“siRNA”) and small hairpin RNA (“shRNA”). “RNA interference” refers to the selective degradation of a sequence-compatible messenger RNA transcript.
As used herein, the term “small interfering RNA” or “siRNA” refers to any small RNA molecule capable of inhibiting or down regulating gene expression by mediating RNA interference in a sequence specific manner. The small RNA can be, for example, about 16 to 21 nucleotides long.
As used herein, the term “shRNA” (small hairpin RNA) refers to an RNA molecule comprising an antisense region, a loop portion and a sense region, wherein the sense region has complementary nucleotides that base pair with the antisense region to form a duplex stem. Following post-transcriptional processing, the small hairpin RNA is converted into a small interfering RNA (siRNA) by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family.
As used herein, the term “antisense oligonucleotide” refers to a synthetic oligonucleotide or oligonucleotide mimetic that is complementary to a DNA or mRNA sequence (e.g., a miRNA).
As used herein, the term “miR-mask” refers to a single stranded antisense oligonucleotide that is complementary to a miRNA binding site in a target mRNA, and that serves to inhibit the binding of miRNA to the mRNA binding site. See, e.g., Xiao, et al. “Novel approaches for gene-specific interference via manipulating actions of microRNAs: examination on the pacemaker channel genes HCN2 and HCN4,” Journal of Cellular Physiology, vol. 212, no. 2, pp. 285-292, 2007, which is incorporated herein in its entirety.
As used herein, the term “miRNA sponge” refers to a synthetic nucleic acid (e.g. a mRNA transcript) that contains multiple tandem-binding sites for a miRNA of interest, and that serves to titrate out the endogenous miRNA of interest, thus inhibiting the binding of the miRNA of interest to its endogenous targets. See, e.g., Ebert et al., “MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells,” Nature Methods, vol. 4, no. 9, pp. 721-726, 2007, which is incorporated herein in its entirety.
As used herein, the term “respiratory chain uncoupling” refers to the dissipation of the mitochondrial inner membrane proton gradient, thereby preventing the synthesis of ATP in the mitochondrion by oxidative phosphorylation.
As used herein, the term “mitochondrial uncoupler” refers to a protein (or the encoding nucleic acid) that can dissipate of the mitochondrial inner membrane proton gradient, thereby preventing the synthesis of ATP in the mitochondrion by oxidative phosphorylation. Exemplary mitochondrial uncouplers include UCP1 and UCP2.
As used herein, the terms “activator” or “repressor” of a mitochondrial uncoupler refers to a protein that serves to upregulate or downregulate, respectively, an activity of a mitochondrial uncoupler.
As used herein, the term “thermogenic regulator” refers to a protein (or the encoding nucleic acid) that regulates thermogenesis either directly or indirectly. The term encompasses mitochondrial uncouplers, and also activators and repressors of mitochondrial uncouplers. Exemplary thermogenic regulators are set forth in Table 1 herein.
As used herein, the term “modulate” refers to increasing or decreasing a parameter. For example, to modulate the activity of a protein that protein's activity could be increased or decreased.
As used herein, the term “activity” of mitochondrial uncoupler or thermogenic regulator refers to any measurable biological activity including, without limitation, mRNA expression, protein expression, or respiratory chain uncoupling.
The “effective amount” of the miRNA agent composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans. In certain embodiments, this physiological condition is obesity.
A “subject” is a vertebrate, including any member of the class Mammalia, including humans, domestic and farm animals, zoo, sports or pet animals, such as mouse, rabbit, pig, sheep, goat, cattle and higher primates.
The term “mammal” refers to any species that is a member of the class mammalia, including rodents, primates, dogs, cats, camelids and ungulates. The term “rodent” refers to any species that is a member of the order rodentia including mice, rats, hamsters, gerbils and rabbits. The term “primate” refers to any species that is a member of the order primates, including monkeys, apes and humans. The term “camelids” refers to any species that is a member of the family camelidae including camels and llamas. The term “ungulates” refers to any species that is a member of the superorder ungulata including cattle, horses and camelids. According to some embodiments, the mammal is a human.
“Treatment”, or “treating” as used herein, is defined as the application or administration of a therapeutic agent (e.g., a miRNA agent or vector or transgene encoding same) to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has the disease or disorder, a symptom of disease or disorder or a predisposition toward a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the predisposition toward disease.
“Pharmacogenomics”, as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers to the study of how a patient's genes determine his or her response to a drug (e.g., a patient's “drug response phenotype”, or “drug response genotype”).
The “effective amount” of the miRNA agent composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans.
II. Thermogenesis and Obesity
In certain embodiments, the invention provides methods for modulating thermogenesis. These methods generally involve contacting cells or tissue with a miRNA agent that modulates activity of at least one mitochondrial uncoupler (e.g., UCP1 and/or UCP2). Such methods and compositions are particularly useful for treating obesity.
Mammalian adipocytes can be categorized into two major categories based on their functional profiles: 1) energy-storing and releasing, lipid-filled white adipocytes (WAT) and; 2) energy-expending and heat producing, mitochondria-rich brown adipocytes (BAT). Until recently, it was believed that BAT underwent rapid involution in early childhood, leaving only vestigial amounts in adults. However, positron-emission tomography (PET) studies performed in humans with the tracer 18F-fluorodeoxyglucose (18F-FDG) demonstrated that: 1) multiple depots of BAT are still present in the cervical, supraclavicular, axillary and paravertebral regions in adult subjects; 2) BAT in adult humans can be rapidly activated by exposure to cold temperatures; 3) there is an inverse correlation between the activity of BAT and age, body-mass index (BMI), the percentage of body fat, fasting plasma glucose level, beta-blocker use and outdoor temperature; and 4) BAT expansion may drive the weight loss associated with catecholamine-producing phaeochromocytomas, whereas beta3-adrenoreceptor polymorphisms leading to a reduction in receptor function have been linked to weight gain and early onset type 2 diabetes mellitus.
Although WAT and BAT are derived from mesenchymal stem cells, they have distinct lineages, with Myf5 (Myogenic Regulatory Factor 5) (shared with skeletal myocyte progenitors), PGC-1alpha and PRDM16 (PR-domain-containing 16) expression distinguishing the brown from white adipocyte precursors. In addition to the classic brown adipocytes, a different type of brown fat cells can be induced in tissues where WAT predominates. The termed “brite” (brown-in-white) adipocyte has been coined and the appearance of brown-like adipocytes within WAT depots is associated with improved metabolic phenotypes. Increasing BAT mass and/or activity offers a degree of protection from obesity. Heat production by BAT is 300 W/g compared to 1 W/g in all other tissues. Relatively limited amounts of BAT would be required to make significant impact on energy balance, since as little as 50 g of BAT would account for 20% of daily energy expenditure. It has been speculated that the estimated 63 g of BAT found in the supraclavicular/paracervical depot of one subject could combust the energy equivalent of 4.1 kg of WAT over 1 year.
Mitochondrial uncoupling proteins (UCP) are members of the family of mitochondrial anion carrier proteins (MACP). UCPs separate oxidative phosphorylation from ATP synthesis with energy dissipated as heat (also referred to as the “mitochondrial proton leak”). UCPs facilitate the transfer of anions from the inner to the outer mitochondrial membrane and the return transfer of protons from the outer to the inner mitochondrial membrane generating heat in the process. UCPs are the primary proteins responsible for thermogenesis and heat dissipation. Uncoupling Protein 1 (UCP1), also named thermogenin, is a BAT specific protein responsible for thermogenesis and heat dissipation. UCP2 is another Uncoupling Protein also expressed in adipocytes. UCPs are part of network of thermogenic regulator proteins (see FIG. 1). Exemplary thermogenic regulators are set forth in Table 1.
Modulation of thermogenic regulators to induce BAT differentiation and/or mitochondrial uncoupling proteins provides a method to induce thermogenesis in a subject and, hence, to treat obesity. However, chemical pharmacologic approaches cannot target these molecules, as they do not belong to the classic ‘target classes’ (kinases, ion channels, G-protein coupled receptors, etc.) that dominate the ‘druggable space’ of traditional drug discovery. Accordingly, the invention provides novel methods and compositions for modulating these thermogenic regulators using miRNA agents.
In certain embodiments, miRNA agents are employed to upregulate the activity of a mitochondrial uncoupler (e.g., the mRNA expression level, protein expression level, or mitochondrial uncoupling activity). Upregulation of a mitochondrial uncoupler can be achieved in several ways. In one embodiment, the miRNA agent directly inhibits the activity of a naturally occurring miRNA that is responsible for downregulation of the activity (e.g., the mRNA expression level, protein expression level) of the mitochondrial uncoupler. In another embodiment, the miRNA agent upregulates the activity (e.g., the mRNA expression level or the protein expression level) of an activator of the mitochondrial uncoupler. This upregulation can be achieved, for example, by directly inhibiting the activity of a naturally occurring miRNA that is responsible for downregulation of the expression of the activator. In yet another embodiment, the miRNA agent downregulates the activity (e.g., the mRNA expression level or the protein expression level) of a repressor of the mitochondrial uncoupler. This downregulation can be achieved, for example, by directly inhibiting the expression of a repressor of a mitochondrial uncoupler using a miRNA agent.
In certain embodiments, miRNA agents are employed that are capable of modulating the activity of multiple thermogenic regulators simultaneously (Pathway-specific miRNA agents as opposed to universal miRNA agents). For example, a single miRNA, agomir or antagomir that binds to multiple thermogenic regulators can be used. This approach is particularly advantageous in that it allows for the modulation of multiple members of an entire signaling pathway using a single miRNA agent.
In certain embodiments, multiple inhibitory miRNA agents (e.g., antagomirs or miR-masks) are employed. These inhibitory miRNA agents can have the same or different miRNA targets.
III. miRNA Agents
In certain embodiments, the invention employs miRNA agents for the modulation of thermogenic regulators (e.g., mitochondrial uncouplers, such as UCP1 and/or UCP2). miRNA agents, suitable for use in the methods disclosed herein, included, without limitation, miRNA, agomirs, antagomirs, miR-masks, miRNA-sponges, siRNA (single- or double-stranded), shRNA, antisense oligonucleotides, ribozymes, or other oligonucleotide mimetics which hybridize to at least a portion of a target nucleic acid and modulate its function.
In certain embodiments, the miRNA agents are miRNA molecules or synthetic derivatives thereof (e.g., agomirs). In one particular embodiment, the miRNA agent is a miRNA. miRNAs are a class of small (e.g., 18-24 nucleotides) non-coding RNAs that exist in a variety of organisms, including mammals, and are conserved in evolution. miRNAs are processed from hairpin precursors of about 70 nucleotides which are derived from primary transcripts through sequential cleavage by the RNAse III enzymes drosha and dicer. Many miRNAs can be encoded in intergenic regions, hosted within introns of pre-mRNAs or within ncRNA genes. Many miRNAs also tend to be clustered and transcribed as polycistrons and often have similar spatial temporal expression patterns. In general, miRNAs are post-transcriptional regulators that bind to complementary sequences on a target gene (mRNA or DNA), resulting in gene silencing by, e.g., translational repression or target degradation. One miRNA can target many different genes simultaneously. Exemplary miRNA molecules for use in the disclosed methods include without limitation: hsa-miR-1-1, hsa-miR-1-2, hsa-miR-7a-g, hsa-miR-105, hsa-miR-1283, hsa-mir-129, hsa-miR-133a-1, hsa-miR-133a-2, hsa-miR-143, hsa-mir-143-5p, hsa-mir-147, hsa-mir-149, hsa-miR-19a-b, hsa-mir-199a, hsa-mir-199b, hsa-mir-200c, hsa-mir-204, hsa-mir-205, hsa-miR-206, hsa-mir-21, hsa-mir-211, hsa-mir-218, hsa-mir-218-1, hsa-mir-218-2, hsa-mir-219-2, hsa-mir-219-2-3p, hsa-mir-22, hsa-mir-22-3p, hsa-mir-22-5p, hsa-mir-24-2, hsa-miR-30a-e, hsa-miR-3177-5p, hsa-mir-325, hsa-mir-331, hsa-mir-331-5p, hsa-miR-3613-3p, hsa-mir-362, hsa-mir-362-5p, hsa-mir-367, hsa-mir-371, hsa-mir-371-5p, hsa-mir-377, hsa-mir-378, hsa-mir-378a-5p, hsa-mir-382, hsa-mir-383, hsa-miR-3658, hsa-mir-422a, hsa-mir-425, hsa-miR-455-3p, hsa-miR-455-5p, hsa-miR-491, hsa-mir-508, hsa-mir-508-5p, hsa-mir-512-1, hsa-mir-512-2, hsa-miR-515-3p, hsa-mir-519e, hsa-miR-520a, hsa-mir-543, hsa-mir-545, hsa-mir-549, hsa-mir-556, hsa-miR-568, hsa-mir-620, hsa-mir-643, hsa-mir-654-3p, hsa-mir-765, hsa-mir-871, hsa-mir-888, hsa-mir-888-3p, hsa-mir-92b, hsa-mir-93, hsa-mir-96, hsa-mir-99a. In other embodiments, exemplary miRNA molecules for use in the disclosed methods miRNA disclosed in Table A and/or Table 8, herein. In one particular embodiment, the miRNA agent is human miR-22, or a functional derivative thereof.
In another particular embodiment, the miRNA agent is an agomir. Agomirs of a particular miRNA can be identified using the screening methods disclosed herein. In one particular embodiment, the agomir is a functional mimetic of human miR-22 (Davidson B L et al., Nat Rev Genet., 12(5):329-340 (2011).
In certain embodiments, the miRNA agents are oligonucleotide or oligonucleotide mimetics that inhibit the activity of one or more miRNA. Examples of such molecules include, without limitation, antagomirs, interfering RNA, antisense oligonucleotides, ribozymes, miRNA sponges and miR-masks. In one particular embodiment, the miRNA agent is an antagomir. In general, antagomirs are chemically modified antisense oligonucleotides that bind to a target miRNA and inhibit miRNA function by preventing binding of the miRNA to its cognate gene target. Antagomirs can include any base modification known in the art. In one particular embodiment, the antagomir inhibits the activity of human miR-22 (van Rooij E et al., Circ Res., 110(3):496-507 (2012); Snead N M et al., Nucleic Acid Ther., 22(3):139-146 (2012); Czech M P et al., Nat Rev Endocrinol., 7(8):473-484 (2011).
In certain embodiments, the miRNA agents are 10 to 50 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies oligonucleotides having antisense portions of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or any range therewithin.
In certain embodiments, the miRNA agents are chimeric oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimeric inhibitory nucleic acids of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures comprise, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5, 220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference in its entirety.
In certain embodiments, the miRNA agents comprise at least one nucleotide modified at the 2′ position of the sugar, most preferably a 2′-O-alkyl, 2′-O-alkyl-O-alkyl or 2′-fluoro-modified nucleotide. In other preferred embodiments, RNA modifications include 2′-fluoro, 2′-amino and 2′ O-methyl modifications on the ribose of pyrimidines, a basic residue or an inverted base at the 3′ end of the RNA. Such modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than 2′-deoxyoligonucleotides against a given target.
A number of nucleotide and nucleoside modifications have been shown to make an oligonucleotide more resistant to nuclease digestion, thereby prolonging in vivo half-life. Specific examples of modified oligonucleotides include those comprising backbones comprising, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH2—NH—O—CH2, CH,  ̃N(CH3) ̃O ̃CH2 (known as a methylene(methylimino) or MMI backbone], CH2—O—N(CH3)—CH2, CH2—N(CH3)—N(CH3)—CH2 and O—N(CH3)—CH2—CH2 backbones, wherein the native phosphodiester backbone is represented as O—P—O—CH1); amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506); peptide nucleic acid (PNA) backbone (wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497), each of which is herein incorporated by reference in its entirety. Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3′ alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321, 131; 5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563, 253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference in its entirety. Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol, 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991, each of which is herein incorporated by reference in its entirety. Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602, the contents of which is incorporated herein in its entirety.
Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference in its entirety.
In certain embodiments, miRNA agents comprise one or more substituted sugar moieties, e.g., one of the following at the 2′ position: OH, SH, SCH3, F, OCN, OCH3OCH3, OCH3O(CH2)nCH3, O(CH2)nNH2 or O(CH2)nCH3 where n is from 1 to about 10; Ci to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2 CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacokinetic/pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy [2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl)] (Martin et al., Helv. Chim. Acta, 1995, 78, 486). Other preferred modifications include 2′-methoxy (2′-O—CH3), 2′-propoxy (2′-OCH2 CH2CH3) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
In certain embodiments, miRNA agents comprise one or more base modifications and/or substitutions. As used herein, “unmodified” or “natural” bases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified bases include, without limitation, bases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2′ deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic bases, e.g., 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and 2,6-diaminopurine. Kornberg, A., DNA Replication, W. H. Freeman & Co., San Francisco, 1980, pp 75-77; Gebeyehu, G., et al. Nucl. Acids Res. 1987, 15:4513). A “universal” base known in the art, e.g., inosine, can also be included. 5-Me-C substitutions can also be included. These have been shown to increase nucleic acid duplex stability by 0.6-1.2OC. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278). Further suitable modified bases are described in U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,750,692, and 5,681,941, each of which is herein incorporated by reference.
It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligonucleotide or even at a single nucleoside within an oligonucleotide.
In certain embodiments, both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds comprise, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.
In certain embodiments, the miRNA agent is linked (covalently or non-covalently) to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include, without limitation, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Mancharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937), each of which is herein incorporated by reference in its entirety. See also U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference in its entirety.
In one particular embodiment, the miRNA agent is linked to (covalently or non-covalently) to a nucleic acid aptamer. Aptamers are synthetic oligonucleotides or peptide molecules that bind to a specific target molecule. Aptamers appropriate for use with the miRNA agents provided herein are described in U.S. Provisional Patent Application No. 61/695,477 filed Aug. 31, 2012 and incorporated by reference herein in its entirety.
Accordingly, in a first aspect, the invention provides an adipocyte-specific miRNA modulator composition comprising i) a targeting moiety that selectively binds to a cellular surface marker on an adipose target cell in a human and ii) a thermogenic miRNA modulator moiety, wherein the targeting moiety facilitates uptake of the miRNA modulatory moiety by the target cell such that the miRNA is capable of targeting a thermogenic pathway and upregulating thermogenesis in the target cell.
In one embodiment, the composition comprises an aptamir comprising an aptamer as the targeting moiety.
In certain embodiments, the aptamers used with the miRNAs disclosed herein specifically bind to cell surface marker proteins on an adipose tissue mesenchymal stem cell (ATMSC), white adipose tissue (WAT) adipocytes and brown adipose tissue (BAT) adipocytes. Cell surface markers for ATMSCs include CD9, CD10, CD13, CD29, CD36, CD44, CD49d, CD54, CD55, CD59, CD73, CD90, CD91, CD105, CD137, CD146, CD166, and HLA-ABC. Cell surface markers for WAT adipocytes include Adiponectin, Caveolin-1, Caveolin-2, CD36 (FAT), CLH-22 (Clathrin Heavy Chain Chr 22), FABP4 (Adipocyte protein 2, aP2), SLC27A1 (FATP1), SLC27A2 (FATP2), GLUT4 (Glucose Transporter 4), Perilipin 2 or Resistin. Cell surface markers for all adipocytes include Neprilysin (CD10), FAT (CD36), Thy-1 (CD90), Low density lipoprotein receptor-related protein 1 (LRP1 or CD91), Caveolin-1, Caveolin-2, Fatty acid binding protein 4 (FABP4), Cell surface glycoprotein MUC18 (CD146), Activated leukocyte cell adhesion molecule (CD166) and Natriuretic peptide receptor A (NPR1). According to other embodiments, the aptamers for use with the miRNAs disclosed herein can also specifically bind to markers of adipose tissue including adiponectin, leptin, resistin, FGF 17, FGF 19, BMP7, PYY, MetAP2, RBP4, endostatin, and angiostatin.
In certain embodiments, the aptamers are selected by the Cell-SELEX technology, which uses whole living cells as the target, whereby aptamers that recognize specific molecules in their native conformation in their natural environment on the surface of intact cells are selected by repeated amplification and binding to living cells. In this cell-based selection, specific cell surface molecules or even unknown membrane receptors can be directly targeted within their native environment, allowing a straightforward enrichment of cell-specific aptamers.
In certain exemplary embodiments, the miRNA modulator is combined with an aptamer to create an “AptamiR” composition. There are many different ways to combine an aptamer and miRNA analog(s) to create an aptamir. They include, for example, aptamer-miRNA analog chimeras, aptamer-splice-switching oligonucleotide chimeras, and aptamer conjugated to nanoparticles or liposomes containing the miRNA analog(s). “Escort Aptamers” may be inserted at the surface of functional polymers, liposomes, and nanoparticles, each of which can carry many miRNA analogs. For instance, the size of thioaptamer-conjugated liposomes is about 120 nm. Nanoparticle approaches have several functional advantages, including, for example, cellular uptake, the ability to cross membranes, and triggered nanoparticle disassembly.
In one embodiment, an aptamiR composition comprises an aptamer that is directly linked or fused to a miRNA modulator. Such aptamiR5 are entirely chemically synthesized, which provides more control over the composition of the conjugate. For instance, the stoichiometry (ratio of miRNA analog per aptamer) and site of attachment can be precisely defined. The linkage portion of the conjugate presents a plurality (2 or more) of nucleophilic and/or electrophilic moieties that serve as the reactive attachment point for the aptamers and miRNA analogs. In addition, the aptamir may further comprise a linker between the aptamer and the miRNA analog. In some embodiments, the linker is a polyalkylene glycol, particularly a polyethylene glycol. In other embodiments, the linker is a liposome, exosome, dendrimer, or comb polymer. Other linkers can mediate the conjugation between the aptamer and the miRNA analog, including a biotinstreptavidin bridge, or a ribonucleic acid. Exemplary non-covalent linkers include linkers formed by base pairing a single stranded portion or overhang of the miRNA moiety and a complementary single-stranded portion or overhang of the aptamer moiety.
In another particular embodiment, an aptamer is combined with a miRNA analog in the form of a liposome-based aptamiR. Liposomes are spherical nanostructures made of a lipid bilayer that can be loaded with pharmaceuticals, such as miRNAs. Furthermore, the liposome surface can be loaded with different substances, such as polyethylene glycol (extending their systemic half life) or molecular recognition moieties like aptamers for specific binding to targeted cells. For example, aptamer-modified liposomes have been developed, with each liposome displaying approximately 250 aptamers tethered to its surface to facilitate target binding. In a preferred embodiment, liposomes are created to encapsulate miRNA analog(s) and display at their surface aptamers that specifically bind with high affinity and specificity to molecules (e.g. lipid transporters) highly expressed at the surface of adipocytes and ATMSCs. The fusion of the liposomes with the targeted cells causes the release of the miRNA analog(s) into the cell cytoplasm, which then alter a specific intra-cellular pathway. Alternatively, stable thioaptamers may be inserted at the surface of liposomes to guide delivery of the liposome miRNA analog(s) load to targeted ATMSCs and adipocytes.
In a further particular embodiment, an aptamer is combined with a miRNA analog in the form of a carrier-based aptamiR. Exemplary carriers include nanoparticles, lipsomes or exosomes. Such carrier-based aptamiR compositions have the capability of delivering a cargo of multiple miRNA modulators to the target cell in a single carrier. To accomplish targeting and accumulation, the carriers are formulated to present the targeting moiety on their external surface so they can react/bind with selected cell surface antigens or receptors on the adipose target cell. As an example, carriers may be created to encapsulate miRNA modulators while displaying at their surface aptamers that specifically bind with high affinity and specificity to molecules (e.g. lipid transporters) highly expressed at the surface of adipocytes and ATMSCs. The internalized exosomes release inside the cell cytoplasm their miRNA analog(s) load, which alters a specific intra-cellular pathway.
In one embodiment, the carrier is an exosome. Exosomes, which originate from late endosomes, are naturally occurring nanoparticles that are specifically loaded with proteins, mRNAs, or miRNAs, and are secreted endogenously by cells. Exosomes are released from host cells, are not cytotoxic, and can transfer information to specific cells based on their composition and the substance in/on the exosome. Because exosomes are particles of approximately 20-100 nm in diameter, the exosomes evade clearance by the mononuclear phagocyte system (which clears circulating particles >100 nm in size), and are very efficiently delivered to target tissues.
Moreover, synthetic exosomes may offer several advantages over other carriers. For example, they may deliver their cargo directly into the cytosol, while their inertness avoids attack and clearance in the extracellular environment. The structural constituents of exosomes may include small molecules responsible for processes like signal transduction, membrane transport, antigen presentation, targeting/adhesion, among many others.
The miRNA agents must be sufficiently complementary to the target mRNA, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect. “Complementary” refers to the capacity for pairing, through hydrogen bonding, between two sequences comprising naturally or non-naturally occurring bases or analogs thereof. For example, if a base at one position of a miRNA agent is capable of hydrogen bonding with a base at the corresponding position of a target nucleic acid sequence, then the bases are considered to be complementary to each other at that position. In certain embodiments, 100% complementarity is not required. In other embodiments, 100% complementarity is required.
miRNA agents for use in the methods disclosed herein can be designed using routine methods. While the specific sequences of certain exemplary target nucleic acid sequences and miRNA agents are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional target segments are readily identifiable by one having ordinary skill in the art in view of this disclosure. Target segments of 5, 6, 7, 8, 9, 10 or more nucleotides in length comprising a stretch of at least five (5) consecutive nucleotides within the seed sequence, or immediately adjacent thereto, are considered to be suitable for targeting a gene. In some embodiments, target segments can include sequences that comprise at least the 5 consecutive nucleotides from the 5′-terminus of one of the seed sequence (the remaining nucleotides being a consecutive stretch of the same RNA beginning immediately upstream of the 5′-terminus of the seed sequence and continuing until the miRNA agent contains about 5 to about 30 nucleotides). In some embodiments, target segments are represented by RNA sequences that comprise at least the 5 consecutive nucleotides from the 3′-terminus of one of the seed sequence (the remaining nucleotides being a consecutive stretch of the same miRNA beginning immediately downstream of the 3′-terminus of the target segment and continuing until the miRNA agent contains about 5 to about 30 nucleotides). One having skill in the art armed with the sequences provided herein will be able, without undue experimentation, to identify further preferred regions to target using miRNA agents. Once one or more target regions, segments or sites have been identified, inhibitory nucleic acid compounds are chosen that are sufficiently complementary to the target, i.e., that hybridize sufficiently well and with sufficient specificity (i.e., do not substantially bind to other non-target nucleic acid sequences), to give the desired effect.
In certain embodiments, miRNA agents used to practice this invention are expressed from a recombinant vector. Suitable recombinant vectors include, without limitation, DNA plasmids, viral vectors or DNA minicircles. Generation of the vector construct can be accomplished using any suitable genetic engineering techniques well known in the art, including, without limitation, the standard techniques of PCR, oligonucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al. Molecular Cloning: A Laboratory Manual. (1989), Coffin et al. (Retroviruses. (1997) and “RNA Viruses: A Practical Approach” (Alan J. Cann, Ed., Oxford University Press, (2000)). As will be apparent to one of ordinary skill in the art, a variety of suitable vectors are available for transferring nucleic acids of the invention into cells. The selection of an appropriate vector to deliver nucleic acids and optimization of the conditions for insertion of the selected expression vector into the cell, are within the scope of one of ordinary skill in the art without the need for undue experimentation. Viral vectors comprise a nucleotide sequence having sequences for the production of recombinant virus in a packaging cell. Viral vectors expressing nucleic acids of the invention can be constructed based on viral backbones including, but not limited to, a retrovirus, lentivirus, adenovirus, adeno-associated virus, pox virus or alphavirus. The recombinant vectors can be delivered as described herein, and persist in target cells (e.g., stable transformants).
In certain embodiments, miRNA agents used to practice this invention are synthesized in vitro using chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; U.S. Pat. No. 4,458,066, each of which is herein incorporated by reference in its entirety.
IV. Methods of Treatment
In one aspect, the invention provides a method of treating obesity in human subject. The method generally comprises administering to the human subject an effective amount of a miRNA agent that modulates activity of at least one thermogenic regulator, (e.g., a mitochondrial uncoupler, such as UCP1 and/or UCP2).
Such methods of treatment may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the target gene molecules of the present invention or target gene modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.
miRNA agents can be tested in an appropriate animal model e.g., an obesity model including ob/ob mice (Lindstrom P., ScientificWorld Journal, 7:666-685 (2007) and db/db mice (Sharma K et al., Am J Physiol Renal Physiol., 284(6):F1138-1144 (2003)). For example, a miRNA agent (or expression vector or transgene encoding same) as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with said agent. Alternatively, a therapeutic agent can be used in an animal model to determine the mechanism of action of such an agent. For example, a miRNA agent can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent can be used in an animal model to determine the mechanism of action of such an agent.
The disclosure also provides a method of inducing pre-adipocytes to differentiate into white adipocytes and white adipocytes into brown adipocytes, comprising administering to a population of pre-adipocytes one or more miRNAs selected from hsa-let-7a agomir, hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-1 antagomir, hsa-miR-19 agomir, hsa-miR-19b agomir, hsa-miR-19b antagomir, hsa-miR-30b agomir and hsa-miR-30b antagomir. In certain embodiments, the induction of pre-adipocytes to differentiate into adipocytes is greater than the differentiation of pre-adipocytes to adipocytes than when pre-adipocytes are exposed to 100 mM rosiglitazone for two days followed by maintenance medium. In certain embodiments, the adipocytes are brown adipocytes. In other embodiments, the adipocytes are white adipocytes.
The disclosure also provides a method for increasing insulin sensitivity in a subject in need thereof comprising administering the subject one or more miRNAs selected from the group consisting of hsa-let-7a agomir, hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-1 antagomir, hsa-miR-19 agomir, hsa-miR-19b agomir, hsa-miR-19b antagomir, hsa-miR-30b agomir and hsa-miR-30b antagomir. In certain embodiments, the subject is a mammal.
The disclosure also provides a method of causing fat loss in a subject in need thereof comprising administering the subject one or more miRNAs selected from the group consisting of hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-19b agomir and hsa-miR-30b agomir. In certain embodiments, the subject is a mammal. In other embodiments, the mammal is a human.
A miRNA agent modified for enhancing uptake into cells (e.g., adipose cells) can be administered at a unit dose less than about 15 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, and less than 200 nmole of miRNA agent (e.g., about 4.4×1016 copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNA silencing agent per kg of bodyweight. The unit dose, for example, can be administered by injection (e.g., intravenous or intramuscular), an inhaled dose, or a topical application. Particularly preferred dosages are less than 2, 1, or 0.1 mg/kg of body weight.
Delivery of a miRNA agent directly to an organ or tissue (e.g., directly to adipose tissue) can be at a dosage on the order of about 0.00001 mg to about 3 mg per organ/tissue, or preferably about 0.0001-0.001 mg per organ/tissue, about 0.03-3.0 mg per organ/tissue, about 0.1-3.0 mg per organ/tissue or about 0.3-3.0 mg per organ/tissue. The dosage can be an amount effective to treat or prevent obesity or to increase insulin sensitivity. In one embodiment, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time. In one embodiment, the effective dose is administered with other traditional therapeutic modalities.
In certain embodiment, a subject is administered an initial dose, and one or more maintenance doses of a miRNA agent. The maintenance dose or doses are generally lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 mg/kg to 1.4 mg/kg of body weight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. The maintenance doses are preferably administered no more than once every 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient. In preferred embodiments the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once every 5 or 8 days. Following treatment, the patient can be monitored for changes in condition, e.g., changes in percentage body fat. The dosage of the compound may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if a decrease in body fat is observed, or if undesired side-effects are observed.
The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stent (e.g., sub-cutaneous, intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable. In one embodiment, a pharmaceutical composition includes a plurality of miRNA agent species. In another embodiment, the miRNA agent species has sequences that are non-overlapping and non-adjacent to another species with respect to a naturally occurring target sequence. In another embodiment, the plurality of miRNA agent species is specific for different naturally occurring target genes. In another embodiment, the miRNA agent is allele specific. In another embodiment, the plurality of miRNA agent species target two or more SNP alleles (e.g., two, three, four, five, six, or more SNP alleles).
Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound of the invention is administered in maintenance doses, ranging from 0.01 mg per kg to 100 mg per kg of body weight (see U.S. Pat. No. 6,107,094).
The concentration or amount of miRNA agent administered will depend on the parameters determined for the agent and the method of administration, e.g. nasal, buccal, or pulmonary. For example, nasal formulations tend to require much lower concentrations of some ingredients in order to avoid irritation or burning of the nasal passages. It is sometimes desirable to dilute an oral formulation up to 10-100 times in order to provide a suitable nasal formulation.
Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a miRNA agent can include a single treatment or, preferably, can include a series of treatments. It will also be appreciated that the effective dosage of a miRNA agent for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein. For example, the subject can be monitored after administering a miRNA agent composition. Based on information from the monitoring, an additional amount of the miRNA agent composition can be administered.
Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In some embodiments, the animal models include transgenic animals that express a human gene, e.g., a gene that produces a target mRNA (e.g., a thermogenic regulator). The transgenic animal can be deficient for the corresponding endogenous mRNA. In another embodiment, the composition for testing includes a miRNA agent that is complementary, at least in an internal region, to a sequence that is conserved between a nucleic acid sequence in the animal model and the target nucleic acid sequence in a human.
Several studies have reported successful mammalian dosing using miRNA agents. For example, Esau C, et al., Cell Metabolism, 3(2): 87-98 (2006) reported dosing of normal mice with intraperitoneal doses of miR-122 antisense oligonucleotide ranging from 12.5 to 75 mg/kg twice weekly for 4 weeks. The mice appeared healthy and normal at the end of treatment, with no loss of body weight or reduced food intake. Plasma transaminase levels were in the normal range (AST 3⁄4 45, ALT 3⁄4 35) for all doses with the exception of the 75 mg/kg dose of miR-122 ASO, which showed a very mild increase in ALT and AST levels. They concluded that 50 mg/kg was an effective, nontoxic dose. Another study by Krutzfeldt J., et al., Nature, 438, 685-689 (2005), injected antagomirs to silence miR-122 in mice using a total dose of 80, 160 or 240 mg per kg body weight. The highest dose resulted in a complete loss of miR-122 signal. In yet another study, locked nucleic acids (“LNAs”) were successfully applied in primates to silence miR-122. Elmen J., et al., (2008) Nature 452, 896-899, report that efficient silencing of miR-122 was achieved in primates by three doses of 10 mg per kg LNA-antimiR, leading to a long-lasting and reversible decrease in total plasma cholesterol without any evidence for LNA-associated toxicities or histopathological changes in the study animals.
In certain embodiments, miRNA agents used to practice this invention are administered through expression from a recombinant vector. Suitable recombinant vectors include, without limitation, DNA plasmids, viral vectors or DNA minicircles. Generation of the vector construct can be accomplished using any suitable genetic engineering techniques well known in the art, including, without limitation, the standard techniques of PCR, oligonucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al. Molecular Cloning: A Laboratory Manual. (1989)), Coffin et al. (Retroviruses. (1997)) and “RNA Viruses: A Practical Approach” (Alan J. Cann, Ed., Oxford University Press, (2000)). As will be apparent to one of ordinary skill in the art, a variety of suitable vectors are available for transferring nucleic acids of the invention into cells. The selection of an appropriate vector to deliver nucleic acids and optimization of the conditions for insertion of the selected expression vector into the cell, are within the scope of one of ordinary skill in the art without the need for undue experimentation. Viral vectors comprise a nucleotide sequence having sequences for the production of recombinant virus in a packaging cell. Viral vectors expressing nucleic acids of the invention can be constructed based on viral backbones including, but not limited to, a retrovirus, lentivirus, adenovirus, adeno-associated virus, pox virus or alphavirus. The recombinant vectors can be delivered as described herein, and persist in target cells (e.g., stable transformants).
miRNA agents may be directly introduced into a cell (e.g., an adipocyte); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be introduced by bathing a cell or organism in a solution containing the nucleic acid. Vascular or extravascular circulation, the blood or lymph system, and the cerebrospinal fluid are sites where the nucleic acid may be introduced.
The miRNA agents of the invention can be introduced using nucleic acid delivery methods known in art including injection of a solution containing the nucleic acid, bombardment by particles covered by the nucleic acid, soaking the cell or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the nucleic acid. Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, and the like. The miRNA agents may be introduced along with other components e.g., compounds that enhance miRNA agent uptake by a cell.
In certain embodiments, the methods described herein include co-administration of miRNA agents with other drugs or pharmaceuticals, e.g., compositions for modulating thermogenesis, compositions for treating diabetes, compositions for treating obesity. Compositions for modulating thermogenesis include beta-3 adrenergic agonists, thyroid hormones, PPARG agonists, leptin, adiponectin, and orexin.
V. Screening Methods
In another aspect, the invention provides a method of screening for a miRNA agent that modulates thermogenesis, decreases obesity, or improves insulin sensitivity. The method generally comprises the steps of: providing an indicator cell; contacting the indicator cell with a test miRNA agent; and determining the expression level and/or cellular activity of at least one thermogenic regulator in the indicator cell in the presence and absence of the miRNA agent, wherein a change in the activity of the thermogenic regulator in the presence of the test miRNA agent identifies the test miRNA agent as a miRNA agent that modulates thermogenesis, decreases obesity, or improves insulin sensitivity. In certain embodiments, the method involves determining an increase the expression level and/or activity of the thermogenic regulator (e.g., UCP1, UCP2). The indicator cell can be a mammalian cell. In certain embodiments, the mammalian cell is a human cell, which comprises at least a portion of a human genome.
Any thermogenic regulator can be assayed in the methods disclosed herein. Exemplary thermogenic regulators are set forth in Table 1. In a preferred embodiment, the thermogenic regulator is a mitochondrial uncoupling protein e.g., UCP1 and/or UCP2.
Any cell in which the activity of a thermogenic regulator can be measured is suitable for use in the methods disclosed herein. Exemplary cells include pre-adipocytes, adipocytes, adipose tissue derived mesenchymal stem cells, hepatocytes, myocytes, or precursors thereof.
Any activity of a thermogenic regulator can be assayed, including, without limitation, mRNA expression level, protein expression level or mitochondrial uncoupling activity of the thermogenic regulator. Methods for determining such activities are well known in the art.
Any miRNA agent can be screened, including, without limitation, miRNA, agomirs, antagomirs, aptamirs, miR-masks, miRNA sponges, siRNA (single- or double-stranded), shRNA, antisense oligonucleotides, ribozymes, or other oligonucleotide mimetics which hybridize to at least a portion of a target nucleic acid and modulate its function.
VI. Pharmaceutical Compositions
In one aspect, the methods disclosed herein can include the administration of pharmaceutical compositions and formulations comprising miRNA agents capable of modulating the activity of at least one thermogenic modulator.
In certain embodiments, the compositions are formulated with a pharmaceutically acceptable carrier. The pharmaceutical compositions and formulations can be administered parenterally, topically, by direct administration into the gastrointestinal tract (e.g., orally or rectally), or by local administration, such as by aerosol or transdermally. The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005.
The miRNA agents can be administered alone or as a component of a pharmaceutical formulation (composition). The compounds may be formulated for administration, in any convenient way for use in human or veterinary medicine. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
Formulations of the compositions of the invention include those suitable for intradermal, inhalation, oral/nasal, topical, parenteral, rectal, and/or intravaginal administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient (e.g., nucleic acid sequences of this invention) which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration, e.g., intradermal or inhalation. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect, e.g., an antigen specific T cell or humoral response.
Pharmaceutical formulations of the invention can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such drugs can contain sweetening agents, flavoring agents, coloring agents and preserving agents. A formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.
Pharmaceutical formulations for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, pills, powder, dragées, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Pharmaceutical preparations for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragée cores. Suitable solid excipients are carbohydrate or protein fillers include, e.g., sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; and gums including arabic and tragacanth; and proteins, e.g., gelatin and collagen. Disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Push-fit capsules can contain active agents mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active agents can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
Aqueous suspensions can contain an active agent (e.g., nucleic acid sequences of the invention) in admixture with excipients suitable for the manufacture of aqueous suspensions, e.g., for aqueous intradermal injections. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin. Formulations can be adjusted for osmolarity.
In certain embodiments, oil-based pharmaceuticals are used for administration of the miRNA agents. Oil-based suspensions can be formulated by suspending an active agent in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. See e.g., U.S. Pat. No. 5,716,928 describing using essential oils or essential oil components for increasing bioavailability and reducing inter- and intra-individual variability of orally administered hydrophobic pharmaceutical compounds (see also U.S. Pat. No. 5,858,401). The oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid. As an example of an injectable oil vehicle, see Minto (1997) J. Pharmacol. Exp. Ther. 281:93-102.
In certain embodiments, the pharmaceutical compositions and formulations are in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent. In alternative embodiments, these injectable oil-in-water emulsions of the invention comprise a paraffin oil, a sorbitan monooleate, an ethoxylated sorbitan monooleate and/or an ethoxylated sorbitan trioleate.
In certain embodiments, the pharmaceutical compositions and formulations are administered by in intranasal, intraocular and intravaginal routes including suppositories, insufflation, powders and aerosol formulations (for examples of steroid inhalants, see e.g., Rohatagi (1995) J. Clin. Pharmacol. 35: 1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol. 75: 107-111). Suppositories formulations can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug. Such materials are cocoa butter and polyethylene glycols.
In certain embodiments, the pharmaceutical compositions and formulations are delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
In certain embodiments, the pharmaceutical compositions and formulations are delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug which slowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed. 7:623-645; as biodegradable and injectable gel formulations, see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oral administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674.
In certain embodiments, the pharmaceutical compositions and formulations are parenterally administered, such as by intravenous (IV) administration or administration into a body cavity or lumen of an organ. These formulations can comprise a solution of active agent dissolved in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter. These formulations may be sterilized by conventional, well known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. For IV administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a suspension in a nontoxic parenterally-acceptable diluent or solvent, such as a solution of 1,3-butanediol. The administration can be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into a blood vessel for a specified period of time).
In certain embodiments, the pharmaceutical compounds and formulations are lyophilized. Stable lyophilized formulations comprising an inhibitory nucleic acid can be made by lyophilizing a solution comprising a pharmaceutical of the invention and a bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose or mixtures thereof. A process for preparing a stable lyophilized formulation can include lyophilizing a solution about 2.5 mg/mL nucleic acid, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. See, e.g., U.S. 20040028670.
In certain embodiments, the pharmaceutical compositions and formulations are delivered by the use of liposomes. By using liposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent into target cells in vivo. See, e.g., U.S. Pat. Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13:293-306; Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46: 1576-1587.
The formulations of the invention can be administered for prophylactic and/or therapeutic treatments. In certain embodiments, for therapeutic applications, compositions are administered to a subject who is need of reduced triglyceride levels, or who is at risk of or has a disorder described herein, in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the disorder or its complications; this can be called a therapeutically effective amount. For example, in certain embodiments, pharmaceutical compositions of the invention are administered in an amount sufficient to treat obesity in a subject.
The amount of pharmaceutical composition adequate to accomplish this is a therapeutically effective dose. The dosage schedule and amounts effective for this use, i.e., the dosing regimen, will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.
The dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84: 1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24: 103-108; Remington: The Science and Practice of Pharmacy, 21st ed., 2005). The state of the art allows the clinician to determine the dosage regimen for each individual patient, active agent and disease or condition treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance to determine the dosage regiment, i.e., dose schedule and dosage levels, administered practicing the methods of the invention are correct and appropriate. Single or multiple administrations of formulations can be given depending on for example: the dosage and frequency as required and tolerated by the patient, the degree and amount of cholesterol homeostasis generated after each administration, and the like. The formulations should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate conditions, diseases or symptoms, e.g., treat obesity.
In certain embodiments, pharmaceutical formulations for oral administration are in a daily amount of between about 1 to 100 or more mg per kilogram of body weight per day. Lower dosages can be used, in contrast to administration orally, into the blood stream, into a body cavity or into a lumen of an organ. Substantially higher dosages can be used in topical or oral administration or administering by powders, spray or inhalation. Actual methods for preparing parenterally or non-parenterally administrable formulations will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, 21st ed., 2005.
VII. Exemplification
The present invention is further illustrated by the following examples which should not be construed as further limiting. The contents of Sequence Listing, figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.
Furthermore, in accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins, eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).
Example 1
In-Silico Analysis of Thermogenic Regulators
Eighty three proteins that are involved in regulation of thermogenesis were selected based upon a critical assessment and review of the available scientific information and our own experimental data. These proteins were categorized as activators or repressors of thermogenesis based upon their functions. These thermogenic regulator proteins are set forth in Table 1.







TABLE 1







Thermogenic regulator proteins.











Name
Entrez Gene ID
Ensembl Gene ID











Activators










1
ALDH1A1
216
ENSG00000165092


2
ANP (NPPA)
4878
ENSG00000175206


3
AZGP1
563
ENSG00000160862


4
BMP7
655
ENSG00000101144


5
BMP8b
656
ENSG00000116985


6
CEBPA
1050
ENSG00000245848


7
CEBPB
1051
ENSG00000172216


8
CEBPD
1052
ENSG00000221869


9
CIDEA
1149
ENSG00000176194


10
COX7A1
1346
ENSG00000161281


11
CRAT
1384
ENSG00000095321


12
CREB1
1385
ENSG00000118260


13
CREBBP
1387
ENSG00000005339


14
CTBP1
1487
ENSG00000159692


15
CTBP2
1488
ENSG00000175029


16
DIO2
1734
ENSG00000211448


17
ELOVL3
83401
ENSG00000119915


18
FGF16
8823
ENSG00000196468


19
FGF19
9965
ENSG00000162344


20
FGF21
26291
ENSG00000105550


21
FNDC5
252995
ENSG00000160097


22
FOXC2
2303
ENSG00000176692


23
GDF3
9573
ENSG00000184344


24
HCRT (OREXIN)
3060
ENSG00000161610


25
HOXC8
3224
ENSG00000037965


26
INSR
3643
ENSG00000171105


27
IRS1
3667
ENSG00000169047


28
KDM3A (JMJD1A)
55818
ENSG00000115548


29
KLF5
688
ENSG00000102554


30
KLF11
8462
ENSG00000172059


31
KLF15
28999
ENSG00000163884


32
LRP6
4040
ENSG00000070018


33
MAPK14
1432
ENSG00000112062


34
MED13
9969
ENSG00000108510


35
NCOA1
8648
ENSG00000084676


36
NCOA2
10499
ENSG00000140396


37
NCOA3
8202
ENSG00000124151


38
NR4A3
8013
ENSG00000119508


39
NRF1
4899
ENSG00000106459


40
PLAC8
51316
ENSG00000145287


41
PPARA
5465
ENSG00000186951


42
PPARD
5467
ENSG00000112033


43
PPARG
5468
ENSG00000132170


44
PPARGC1A
10891
ENSG00000109819


45
PPARGC1B
133522
ENSG00000155846


46
PRDM16
63976
ENSG00000142611


47
PRDX3
10935
ENSG00000165672


48
PRKAA1 (AMPKA1)
5562
ENSG00000132356


49
PRKAA2 (AMPKA2)
5563
ENSG00000162409


50
PRKACA
5566
ENSG00000072062


51
PRKACB
5567
ENSG00000142875


52
PRKAR1A
5573
ENSG00000108946


53
SIRT1
23411
ENSG00000096717


54
SIRT3
23410
ENSG00000142082


55
SLC27A2 (FATP2)
11001
ENSG00000140284


56
SREBF1
6720
ENSG00000072310


58
SREBF2
6721
ENSG00000198911


58
STAT5A
6776
ENSG00000126561


59
TRPM8
79054
ENSG00000144481


60
UCP1 (SLC25A7)
7350
ENSG00000109424


61
UCP2 (SLC25A8)
7351
ENSG00000175567


62
UCP3 (SLC25A9)
7352
ENSG00000175564







Repressors










1
ATG7
10533
ENSG00000197548


2
BMP2
650
ENSG00000125845


3
BMP4
652
ENSG00000125378


4
CIDEC
63924
ENSG00000187288


5
CTNNB1
1499
ENSG00000168036


6
DLK1 (Pref-1)
8788
ENSG00000185559


7
E2F4 (p107)
1874
ENSG00000205250


8
EIF4EBP1
1978
ENSG00000187840


9
ESRRA (NR3B1)
2101
ENSG00000173153


10
IKBKE
9641
ENSG00000143466


11
NR1H3 (LXRA)
10062
ENSG00000025434


12
NRIP1 (RIP140)
8204
ENSG00000180530


13
RB1 (pRb)
5925
ENSG00000139687


14
NR0B2 (SHP)
8431
ENSG00000131910


15
RPS6KB1
6198
ENSG00000108443


16
RUNX1T1
862
ENSG00000079102


17
RUNX2
860
ENSG00000124813


18
TNFRSF1A
7132
ENSG00000067182


19
TWIST1
7291
ENSG00000122691


20
WNT5A
7474
ENSG00000114251


21
WNT10B
7480
ENSG00000169884









The STRING 9.0 database of known and predicted protein interactions (string-db.org/) was used to test these 83 candidate molecules. The interactions include direct (physical) and indirect (functional) associations; they are derived from four sources: genomic context; high-throughput experiments; co-expression; and previous knowledge. STRING quantitatively integrates interaction data from these sources for a large number of organisms, and transfers information between these organisms where applicable. The database currently covers 5,214,234 proteins from 1,133 organisms. As an example, the relationships between the 83 thermogenic regulator molecules were centered around UCP1, and molecules having direct and indirect connections with UCP1 could be distinguished using the highest confidence score of 0.90. This relationship is set forth in schematic form in FIG. 1A. From this analysis, it was discovered that nine molecules (CEBPB, CIDEA, KDM3A, NRIP1, PRDM16, PPARG, PPARGC1A, PPKAA2, and UCP2) are directly linked to UCP1, whereas many more molecules are connected to UCP1 on a second or higher degree order.
When the degree of confidence was set to high with a score of 0.70, eight additional proteins were found to be directly linked to UCP1 (AZGP1, DIO2, KLF11, KLF15, NR1H3, PPARA, PPARD, and PPARGC1B), FIG. 1B.
Similarly, the interactions among these 83 thermogenic regulator molecules were independently assessed using other software programs. The interactions predicted by the Ingenuity Pathway Analysis (IPA) Software program (www.ingenuity.com) are shown in FIG. 2A (UCP1 in yellow, activators in green and repressors in purple). The interactions predicted by the Reactome Functional Interaction (Reactome IF) Software program (http://wiki.reactome.org) are shown in FIG. 2B (UCP1 in yellow, activators in green and repressors in purple). The IPA and Reactome IF networks differ from the one set forth in FIG. 1, obtained with the STRING program. It is not surprising that the results of these algorithms are different because they rely on different predefined parameters, sources of information and selection criteria.
Example 2
In-Silico Selection of Relevant miRNA Targets
To select thermogenic regulators suitable as targets for miRNA agents, several internet-based resources were employed to match miRNAs and their targets (the “micronome”). Exemplary tools are set forth in Table 2A.







TABLE 2A







Exemplary bioinformatics tools used to select miRNAs and their targets.









Field & Name
Function
Web Address





Integrated Data




Mining (8)


BioCarta
Catalogs and summarizes important
biocarta.com



resources providing information for



over 120,000 genes from multiple



species. Find both classical



pathways as well as current



suggestions for new pathways


Database for
Integrated biological
david.abcc.ncifcrf.gov/home.jsp


Annotation,
knowledgebase and analytic tools


Visualization and
aimed at systematically extracting


Integrated
biological meaning from large


Discovery (DAVID)
gene/protein lists


GeneOntology
Standardizing the representation of
geneontology.org/



gene and gene product attributes



across species and databases


Gene Set
Computational method that
broadinstitute.org/gsea/index.jsp


Enrichment
determines whether an a priori


Analysis (GSEA)
defined set of genes shows



statistically significant, concordant



differences between two biological



states (e.g. phenotypes).


KEGG
Kyoto Encyclopedia of Genes and
genome.jp/kegg/



Genomes


PubGene
Connecting up-to-date information
pubgene.org/



on genes and related terms


Reactome
An open-source, open access,
reactome.org/ReactomeGWT/entry



manually curated and peer-
point.html



reviewed pathway database.


STRING
Database of known and predicted
string-db.org/



protein interactions; direct



(physical) and indirect (functional)



associations


miRNA Mining


& Mapping (8)


deepBase
Platform for annotating and
deepbase.sysu.edu.cn/



discovering small and long ncRNAs



(microRNAs, siRNAs, piRNAs . . .)


Human
Contains miRNA names, disease
202.38.126.151/hmdd/mirna/md/


microRNA
names, dysfunction evidences, and


disease database
the literature PubMed ID


(HMDD)


miRBase V19
Searchable database of published
mirbase.org/



miRNA sequences and annotation


miRGen 2.0
Database of microRNA genomic
diana.cslab.ece.ntua.gr/mirgen/



information and regulation


miRNAMap
Experimentally verified microRNAs
mirnamap.mbc.nctu.edu.tw/



Shows tissue expression profile


miRSel
Automated extraction of
services.bio.ifi.lmu.de/mirsel/



associations between microRNAs



and genes from the biomedical



literature


miRStart
Database of human microRNA
mirstart.mbc.nctu.edu.tw/home.php



TSSs (transcription start sites)


miR2Disease
A manually curated database
mir2disease.org



providing a comprehensive resource



of miRNA deregulation in various



human diseases


miRNA Targets


& Expression (21)


DIANA-microT
Algorithm based on several
diana.cslab.ece.ntua.gr/microT/


3.0
parameters calculated individually



for each microRNA and it combines



conserved and non-conserved



microRNA recognition elements



into a final prediction score.


DIANA-
Algorithm that can identify
diana.cslab.ece.ntua.gr/hexamers/


mirExTra
microRNA effects to the Expression



levels of protein-coding transcripts,



based on the frequency of six



nucleotide long motifs in the 3′UTR



sequences of genes.


GSEA Molecular
Gene sets that contain genes sharing
broadinstitute.org/gsea/index.jsp


Signatures
a 3′-UTR microRNA binding motif


Database v3.0
(n = 221)


MicroCosm
Computationally predicted targets
ebi.ac.uk/enright-


Targets
for microRNAs across many
srv/microcosm/cgi-



species. The miRNA sequences are
bin/targets/v5/download.pl



obtained from the miRBase



Sequence database and most



genomic sequence from EnsEMBL


MicroInspector
A scanning software for detection
bioinfo.uni-



of miRNA binding sites using
plovdiv.bg/microinspector/



hybridization temperature and free



energy cut-off value


microRNA.org
Predicted microRNA targets &
microrna.org/microrna/home.do


(ex. miRanda)
target downregulation scores.



Experimentally observed



expression patterns.


miRDB
Online database for miRNA target
mirdb.org/miRDB/



prediction and functional



annotations in animals by a new



bioinformatics tool analyzing



thousands of genes impacted by



miRNAs with an SVM learning



machine.


miRTarBase
Has accumulated more than three
mirtarbase.mbc.nctu.edu.tw/index.h



thousand miRNA-target
tml



interactions (MTIs), which are



collected by manually surveying



pertinent literature


miRTar.Human
An integrated web server for
mirtar.mbc.nctu.edu.tw/human/dow



identifying miRNA-target
nload.php



interactions. Identifies the biological



functions and regulatory



relationships between a group of



known/putative miRNAs and



protein coding genes. It also



provides perspective of information



on the miRNA targets on



alternatively spliced transcripts in



human


miRvestigator
Takes as input a list of co-expressed
mirvestigator.systemsbiology.net/



genes and will return the most likely



miRNA regulating these genes. It



does this by searching for an over-



represented sequence motif in the



3′UTRs of the genes using Weeder



and then comparing this to the



miRNA seed sequences in miRBase



using our custom built



miRvestigator hidden Markov



model (HMM)


mirZ
A server that provides statistical
mirz.unibas.ch/E1MMo2/



analysis and data mining tools



operating on up-to-date databases



of sequencing-based miRNA



expression profiles and of predicted



miRNA target sites


MultiMiTar
A Support Vector Machine (SVM)
isical.ac.in/~bioinfo_miu/multimita



based classifier integrated with a
r.htm



multiobjective metaheuristic based



feature selection technique.


PhenomiR
Provides information about
mips.helmholtz-



differentially regulated miRNA
muenchen.de/phenomir/index.gsp



expression in diseases and other



biological processes. The content of



PhenomiR is completely generated



by manual curation of experienced



annotators. Data was extracted from



more than 365 scientific articles and



resulted in more than 632 database



entries as of February 2011


PicTar
Algorithm for the identification of
pictar.mdc-berlin.de/



microRNA targets. This searchable



website provides details (3′ UTR



alignments with predicted sites,



links to various public databases,



etc.)


PITA
Incorporates the role of target-site
genie.weizmann.ac.il/pubs/mir07/m



accessibility, as determined by base-
ir07_data.html



pairing interactions within the



mRNA, in microRNA target



recognition.


RepTar
Database of miRNA target
bioinformatics.ekmd.huji.ac.il/repta



predictions, based on an algorithm
r/



that is independent of evolutionary



conservation considerations and is



not limited to seed pairing sites.


RNAhybrid
A tool for finding the minimum free
bibiserv.techfak.uni-



energy hybridisation of a long and a
bielefeld.de/rnahybrid/



short RNA. The hybridisation is



performed in a kind of domain



mode, ie. the short sequence is



hybridised to the best fitting part of



the long one.


RNA22
First finds putative microRNA
cbcsrv.watson.ibm.com/rna22.html



binding sites in the sequence of



interest, then identifies the targeted



microRNA (IBM).


Sylamer
A system for finding significantly
ebi.ac.uk/enright/sylamer/



over or under-represented words in



sequences according to a sorted



gene list. It is used to find



significant enrichment or depletion



of microRNA or siRNA seed



sequences from microarray



expression data.


TarBase 6.0
Database of experimentally
diana.cslab.ece.ntua.gr/DianaTools



supported microRNA targets.
New/index.php?r=tarbase/index


TargetScanHuman
Predicts biological targets of
targetscan.org/


6.2
miRNAs by searching for the



presence of conserved 8mer and



7mer sites that match the seed



region of each miRNA, using 6



features: site-type contribution, 3′



pairing contribution, local AU



contribution, position contribution,



TA (target site abundance)



contribution, SPS (seed-pairing



stability) contribution.


Integrated


miRNA Targets


& Expression


Tools (13)


GOmir
Integrates the predicted target genes
bioacademy.gr/bioinformatics/proje



from TargetScan, miRanda,
cts/GOmir/



RNAhybrid and PicTar



computational tools and also



providing a full gene description



and functional analysis for each



target gene.


MAMI
Compiles predictions from five
mami.med.harvard.edu/


(MetaMiR: Target
different miRNA target prediction


Inference)
algorithms (TargetScanS, miRanda,



microT, miRtarget, and



picTar).


mimiRNA
Allows the visualization of miRNA
mimirna.centenary.org.au/mep/for



expression levels in 188 different
mulaire.html



tissue or cell types, provides a



robust statistical method for



discovering functional interactions



between miRNAs and mRNA



genes. Uses a novel sample



classification algorithm, ExParser,



that allows mimiRNA to



automatically classify imported



experiments with minimal curation


MMIA
Integrates the predicted target genes
147.46.15.115/MMIA/index.html


(microRNA and
from TargetScan, PicTar, PITA


mRNA Integrated


Analysis)


mirDIP
Integrates twelve microRNA
ophid.utoronto.ca/mirDIP/



prediction datasets from six



microRNA prediction databases,



allowing users to customize their



microRNA target searches.



Combining microRNA predictions



allows users to obtain more robust



target predictions, giving you more



confidence in your microRNA



targets.


miRGator V3.0
Integrated database of miRNA-
mirgator.kobic.re.kr



associated gene expression, target



prediction, disease association and



genomic annotation, using



mirBridge, miRanda, PITA and



TargetScan. Now includes 73 deep



sequencing datasets on human



samples from GEO, SRA, and



TCGA archives


miRecords
Integrates the predictions form
mirecords.biolead.org/



DIANA-microT, MicroInspector,



miRanda, mirTarget2, miTarget,



NBmiRTar, PicTar, PITA, rna22,



RNAhybrid, TargetScan/TargetScanS


MIRNA-
Automatically extracts miRNAs
ikp-


DISTILLER
predicted to interact with a given
stuttgart.de/content/language1/html/



set of target genes from several
10415.asp



selectable public databases.


MiRonTop
Online java web tool that integrates
microarray.fr:8080/miRonTop/inde



DNA microarrays or high-
x



throughput sequencing data to



identify the potential implication of



miRNAs on a specific biological



system. It allows a rapid



characterization of the most



pertinent mRNA targets according



to several existing miRNA target



prediction approaches (Mirbase,



miRanda, exact seed, TargetScan or



PicTar)


miRror
Integrates predictions from a dozen
proto.cs.huji.ac.il/mirror



of miRNA resources that are based



on complementary algorithms into



a unified statistical framework.


miRSystem
Database which integrates 7
mirsystem.cgm.ntu.edu.tw/



miRNA target gene prediction



programs: DIANA, miRanda,



miRBridge, PicTar, PITA, rna22,



and TargetScan.


miRWalk
Comprehensive database that
ma.uni-



provides information on miRNA on
heidelberg.de/apps/zmf/mirwalk/in



their predicted as well as validated
dex.html



binding sites on their target genes.


StarBase
Public platform for decoding
starbase.sysu.edu.cn/index.php



microRNA-target and protein-RNA



interaction maps from CLIP-Seq



(HITS-CLIP, PAR-CLIP) and



degradome sequencing



(Degradome-Seq, PARE) data.


miRNA


Secondary


Structure (5)


OligoWalk
An online server calculating
rna.urmc.rochester.edu/cgi-



thermodynamic features of sense-
bin/server_exe/oligowalk/oligowalk



antisense hybridization. It predicts
_form.cgi



the free energy changes of



oligonucleotides binding to a target



RNA. It can be used to design



efficient siRNA targeting a given



mRNA sequence.


PicTar RNA
The BiBiServ Tool section offers
www.pictar.org/


Studio
bioinformatics tools for a large



variety of tasks, including RNA



studio


RNA2D
Suite of programs for discovering
protein3d.ncifcrf.gov/shuyun/rna2d.



structural features in RNAs.
html


Vienna RNA
RNA Secondary Structure
tbi.univie.ac.at/ivo/RNA/


Package
Prediction and Comparison.


Whitehead siRNA
Helps select siRNAs to knock down
jura.wi.mit.edu/bioc/siRNAext/


algorithm
your gene of interest


Network


Searches &


Analyses (8)


ARIADNE
Pathway analysis software helping
ariadnegenomics.com/products/path


Pathway Studio
to:
way-studio/



Interpret gene expression and



other high throughput data



Build, expand and analyze



pathways



Find relationships among genes,



proteins, cell processes and



diseases



Draw publication-quality diagrams


Cytoscape
Open source bioinformatics
cytoscape.org/



software platform for visualizing



molecular interaction networks and



biological pathways and integrating



these networks with annotations,



gene expression profiles and other



state data.


Database for
Integrated biological
david.abcc.ncifcrf.gov/home.jsp


Annotation,
knowledgebase and analytic tools


Visualization and
aimed at systematically extracting


Integrated
biological meaning from large


Discovery
gene/protein lists


(DAVID)


Genego MetaCore
An integrated knowledge database
genego.com/metacore.php



and software suite for pathway



analysis of experimental data and



gene lists based on a proprietary



manually curated database of human



protein-protein, protein-DNA and



protein compound interactions,



metabolic and signaling pathways.


Ingenuity Systems
To understand biology at multiple
ingenuity.com/products/IPA/micro


IPA (Ingenuity
levels by integrating data from a
RNA.html


Pathway Analysis)
variety of experimental platforms



and providing insight into the



molecular and chemical



interactions, cellular phenotypes,



and disease processes.


MATISSE
A program for detection of
acgt.cs.tau.ac.il/matisse/


(Module Analysis
functional modules using interaction


via Topology of
networks and expression data


Interactions and


Similarity SEts)


MIR@NT@N
a framework integrating
mironton.uni.lu



transcription factors, microRNAs



and their targets to identify sub-



network motifs in a meta-regulation



network model


NAViGaTOR
Network Analysis, Visualization, &
ophid.utoronto.ca/navigator/index.h



Graphing TORonto is a software
tml



package for visualizing and



analyzing protein-protein interaction



networks.


Molecular


Visualization (4)


Foldit
Multiplayer online game that enlists
fold.it/portal/info/science



players worldwide to solve difficult



protein-structure prediction



problems.


PyMOL
A user-sponsored molecular
.pymol.org/



visualization system on an open-



source foundation.


Qlucore Omics
To examine and analyze data from
qlucore.com/ProdOverviewmiRNA


Explorer
miRNA experiments.
.aspx


WebMol
Displays and analyzes structural
cmpharm.ucsf.edu/cgi-



information contained in the
bin/webmol.pl



Brookhaven Protein Data Bank



(PDB). It can be run as an applet or



as a stand-alone application.


Information


Integration (1)


TIBCO Spotfire
Comprehensive software platform



that allows customers to analyze



data, using predictive and complex



statistics in the analysis









Specifically, these tools were used to perform: 1) Integrated Data Mining (8 tools); 2) miRNA Mining and Mapping (6 tools); 3) miRNA Target Targets and Expression (21 tools); 4) Integrated miRNA Targets and Expression (13 tools); 5) miRNA Secondary Structure Prediction and Comparison (5 tools); 6) Network Searches and Analyses (8 tools); 7) Molecular Visualization (4 tools); and 8) Information Integration and Exploitation (1 tool).
A single gene target can be controlled by several miRNAs whereas a single miRNA can control several gene targets. Sophisticated bioinformatics resources have been developed to select the most relevant miRNAs to target diseases (Gallagher I J, et al. Genome medicine. 2010; Fujiki K, et al. BMC Biol. 2009; Okada Y, et al., J Androl. 2010; Hao T, et al., Mol. Biosyst. 2012; Hao T, et al., Mol. Biosyst. 2012). However, the results of these algorithms are acutely dependent on predefined parameters and the degree of convergence between these algorithms is rather limited. Therefore, there is a need to develop better performing bioinformatics tools with improved sensitivity, specificity and selectivity for the identification of miRNA/target relationships.
The interactions between miRNAs and their targets go beyond the original description of miRNAs as post-transcriptional regulators whose seed region of the driver strand (5′ bases 2-7) bind to complementary sequences in the 3′ UTR region of target mRNAs, usually resulting in translational repression or target degradation and gene silencing. The interactions can also involve various regions of the driver or passenger strands of the miRNAs as well as the 5′UTR, promoter, and coding regions of the mRNAs.
Upon analysis of the available data, it was decided to favor pathway-specific miRNAs which target multiple genes within one discrete signaling pathway, rather than universal miRNAs which are involved in many signaling pathways, functions or processes. Using 34 publicly available Internet tools predicting miRNA targets, specific huma miRNAs were searched for that could potentially modulate several targets among the 83 thermogenic regulator molecules (which include 36 Transcription Factors) selected in Example 1.
Several paradigms were considered:
a) A One microRNA-Multiple mRNAs Pathway-Specific Paradigm.
A. The methylation state of histones can be dynamically regulated by histone methyltransferases and demethylases. The human lysine (K)-specific demethylase 3A (KDM3A) is critically important in regulating the expression of metabolic genes. Its loss of function results in obesity and hyperlipidemia in mice. Beta-adrenergic stimulation of KDM3A binding to the PPAR responsive element (PPRE) of the UCP1 gene not only decreases levels of H3K9me2 (dimethylation of lysine 9 of histone H3) at the PPRE, but also facilitates the recruitment of PPARG and RXRA and their co-activators PPARGC1A, CREBBP and NCOA1 to the PPRE. The interrogation of the TargetScan Human database (release 6.0) revealed that the human KDM3A 3′ UTR 29-35 region is a conserved target for hsa-miR-22. Several other miRNA Targets Databases also confirmed this match between hsa-miR-22 and KDM3A. Therefore, increased production of the demethylase KDM3A by an hsa-miR-22 antagomir should lead to demethylation of the UCP1 gene promoter region, thus facilitating binding of several regulatory elements and increased UCP1 production.
In addition, we used the 34 miRNA Targets and Expression tools (Table 2B) to identify the mRNA targets of a given miRNA.







TABLE 2B





Bioinformatics tools used to select miRNAs and their targets.




































1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18





DIANA-microT 3.0
1
X






✓












DIANA-mirExTra
2

X


















GOmir
3


X




✓












GSEA MSD v3.0
4



X
















MAMI
5
✓



X
✓

✓












MicroCosm Targets
6





X














MicroInspector
7






X













microRNA.org
8







X












mimiRNA
9







✓
X











MMIA
10









X










miRDB
11










X









mirDIP
12
✓




✓

✓



X








miRGator v3.0
13





✓

✓


✓

X
✓






miRecords
14
✓





✓
✓





X






MiRNA Distiller
15





✓




✓



X





MiRonTop
16





✓

✓







X




miRror
17
✓




✓

✓


✓





X



miRSystem
18
✓






✓









X


miRTarBase
19




















miRTar · Human
20




















miRvestigator
21




















miRWalk
22
✓






✓


✓









mirZ
23




















MultiMiTar
24




















PhenomiR
25




















PicTar
26




















PITA
27




















RepTar
28




















RNA22
29




















RNAhybrid
30




















StarBase
31







✓












Sylamer
32




















TarBase 6.0
33




















TargetScanHuman
34






















5




4
1
10


3
































19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34







DIANA-microT 3.0
1




















DIANA-mirExTra
2




















GOmir
3







✓



✓



✓
4



GSEA MSD v3.0
4




















MAMI
5







✓







✓
5



MicroCosm Targets
6




















MicroInspector
7




















microRNA.org
8




















mimiRNA
9







✓


✓




✓
4



MMIA
10







✓
✓






✓
3



miRDB
11




















mirDIP
12







✓
✓

✓




✓
7



miRGator v3.0
13
✓






✓
✓





✓
✓
9



miRecords
14







✓
✓

✓
✓



✓
8



MiRNA Distiller
15















✓
3



MiRonTop
16







✓







✓
4



miRror
17




✓


✓
✓

✓




✓
9



miRSystem
18







✓
✓

✓




✓
7



miRTarBase
19
X



















miRTar · Human
20

X


















miRvestigator
21


X

















miRWalk
22



X



✓
✓

✓
✓



✓
8



mirZ
23




X















MultiMiTar
24





X














PhenomiR
25






X













PicTar
26







X












PITA
27








X











RepTar
28









X










RNA22
29










X









RNAhybrid
30











X








StarBase
31







✓
✓

✓

X


✓
5



Sylamer
32













X






TarBase 6.0
33














X





TargetScanHuman
34















X










1


10
8

7
3



12






Meta Tools in bold (13)


Engines called by Meta Tools in Italics (11)






Applying the above in silico strategy, it was discovered that hsa-miR-22-3p and hsa-miR-22-5p interact respectively with a total of 42 and 8 of the chosen 83 thermogenic targets. This data is set forth in Table 3.







TABLE 3







Thermogenic regulators identified as predicted and/or validated targets 


for hsa-miR-22-3p.














ALDH1A1
DIO2
NCOA1
PRKAA1
STAT5A


BMP4
FGF19
NPPA
PRKACA
TNFRSF1A


BMP7
FGF21
NRF1
PRKACB
TRPM8


CEBPA
FOXC2
NRIP1
PRKAR1A
UCP2


CEBPD
INSR
PPARA
RUNX1T1
WNT10B


CIDEC
KDM3A
PPARGC1A
RUNX2
WNT5A


CREB1
KLF11
PPARGC1B
SIRT1



CREBBP
LRP6
PRDM16
SREBF1



CTNNB1
MAPK14
PRDX3
SREBF2










Thermogenic regulators identified as predicted and/or validated targets 


for hsa-miR-22-5p

















BMP7
DIO2
FNDC5
IKBKE
INSR
MAPK14
NR1H3
PPARA









B. We also utilized the 34 miRNA Targets and Expression tools (Table 2B) to look for potential relations between any of the adipocyte 536 miRNAs (Table A) and the 83 thermogenic targets (Table 1).
It appears that many adipocyte miRNAs interact (prediction and/or validation) with at least one of the 83 thermogenic targets. For example, miR-17-3p and hsa-miR-17-5p interact respectively with a total of 23 and 65 of the chosen thermogenic 83 targets. This data is set forth in Table 4.







TABLE 4







Thermogenic regulators identified as predicted


and/or validated targets for hsa-miR-17-3p.











ATG7
CTBP2
KLF11
PPARD
TNFRSF1A


BMP2
E2F4
MAPK14
PRDM16
TWIST1


BMP4
FGF19
NCOA3
RB1
WNT10B


CEBPB
IKBKE
PLAC8
RUNX1T1


CREB1
IRS1
PPARA
STAT5A







Thermogenic regulators identified as predicted


and/or validated targets for hsa-miR-17-5p











ALDH1A1
CREB2
IKBKE
NRIP1
RB1


ATG7
CTNNB1
INSR
PLAC8
RPS6KB1


BMP2
CTBP1
IRS1
PPARA
RUNX1T1


BMP4
CTBP2
KLF11
PPARD
RUNX2


BMP7
DIO2
MAPK14
PPARG
SIRT1


BMP8b
ELOVL3
MED13
PPARGC1A
SIRT3


CEBPA
FGF19
NCOA1
PPARGC1B
SREBF1


CEBPB
FGF21
NCOA2
PRDX3
STAT5A


CEBPD
FNDC5
NCOA3
PRKAA1
TNFRSF1A


CIDEC
FOXC2
NPPA
PRKAA2
TWIST1


COX7A1
GDF3
NR1H3
PRKACA
UCP1


CRAT
HCRT
NR4A3
PRKACB
UCP3


CREB1
HOXC8
NRF1
PRKAR1A
WNT5A









Once the lists of miRNAs of interest and their mRNA targets were produced, the following filters were applied to refine the results:
Parameters
1 Expression of miRNAs in tissue/cell of interest
2 Number of algorithms predicting one miRNA for a given gene or set of genes
3 Score/percent from algorithms
4 Number of preferred genes targeted by one miRNA
5 Number of binding sites in a target gene for one miRNA
6 Number of binding sites in a target gene for several miRNAs
7 Over-representation of one miRNA seed complementary sequence among target genes (miRvestigator)
8 Validated miRNA-mRNA target couples
9 Genomic location of miRNA binding site (5′UTR-Promoter-CDS-3′UTR)
10 Intronic location of miRNA
11 Clustering of miRNAs
12 Abundance of miRNA in specific tissue/cell of interest
Applying the above parameters, it was discovered that 229 miRNAs met at least two of these criteria. This data is set forth in Table 5.







TABLE 5





Ranking of miRNAs according to selection critria.



















hsa-miR-20b-5p
hsa-let-7c
hsa-miR-30d-5p



hsa-miR-27b-3p
hsa-let-7d-5p
hsa-miR-424-5p



hsa-miR-103a-3p
hsa-miR-141-3p
hsa-miR-454-3p



hsa-miR-22-3p
hsa-miR-183-5p
hsa-miR-545-3p



hsa-miR-34a-5p
hsa-miR-19a-3p
hsa-miR-485-5p



hsa-miR-130b-3p
hsa-miR-196a-5p
hsa-miR-335-5p



hsa-miR-132-3p
hsa-miR-30b-5p
hsa-miR-133a



hsa-miR-181b-5p
hsa-miR-378a-3p
hsa-miR-222-3p



hsa-miR-211-5p
hsa-miR-302c-5p
hsa-miR-494



hsa-miR-148b-3p
hsa-miR-30e-5p
hsa-miR-498



hsa-miR-17-5p
hsa-miR-130a-3p
hsa-miR-513a-5p



hsa-miR-182-5p
hsa-let-7e-5p
hsa-miR-92a-3p



hsa-miR-20a-5p
hsa-miR-216a-5p
hsa-miR-495-3p



hsa-miR-27a-3p
hsa-miR-450a-5p
hsa-miR-503-5p



hsa-miR-301a-3p
hsa-let-7d-3p
hsa-miR-539-5p



hsa-miR-204-5p
hsa-miR-26b-5p
hsa-miR-16-2-3p



hsa-miR-143-3p
hsa-miR-181c-5p
hsa-miR-302b-5p



hsa-miR-1
hsa-miR-186-5p
hsa-miR-425-3p



hsa-miR-9-5p
hsa-miR-519c-3p
hsa-miR-99a-3p



hsa-miR-30a-5p
hsa-let-7b-5p
hsa-let-7a-3p



hsa-miR-138-5p
hsa-miR-10b-5p
hsa-miR-126-3p



hsa-miR-217
hsa-miR-125b-5p
hsa-miR-20a-3p



hsa-miR-19b-3p
hsa-miR-134
hsa-miR-499a-5p



hsa-miR-382-5p
hsa-miR-137
hsa-let-7g-5p



hsa-miR-106a-5p
hsa-miR-150-5p
hsa-miR-152



hsa-miR-107
hsa-miR-153
hsa-miR-26a-5p



hsa-miR-135a-5p
hsa-miR-15b-5p
hsa-miR-124-3p



hsa-miR-93-5p
hsa-miR-16-5p
hsa-miR-203a



hsa-miR-21-5p
hsa-miR-195-5p
hsa-miR-24-3p



hsa-miR-515-3p
hsa-miR-196b-5p
hsa-miR-301b



hsa-miR-106b-3p
hsa-miR-23a-3p
hsa-miR-590-3p



hsa-miR-125a-5p
hsa-miR-29c-3p
hsa-miR-1179



hsa-miR-148a-3p
hsa-miR-373-3p
hsa-miR-325



hsa-miR-155-5p
hsa-miR-7-5p
hsa-miR-552



hsa-miR-181a-5p
hsa-miR-214-3p
hsa-miR-185-5p



hsa-miR-519d
hsa-miR-421
hsa-miR-455-3p



hsa-miR-96-5p
hsa-miR-15a-5p
hsa-miR-583



hsa-miR-212-3p
hsa-miR-193b-3p
hsa-miR-122-5p



hsa-miR-29a-3p
hsa-miR-194-5p
hsa-miR-1305



hsa-miR-98-5p
hsa-miR-223-3p
hsa-miR-139-5p



hsa-miR-146a-5p
hsa-miR-30c-5p
hsa-miR-224-3p



hsa-miR-18a-5p
hsa-miR-335-3p
hsa-miR-24-1-5p



hsa-miR-18b-5p
hsa-miR-374a-5p
hsa-miR-24-2-5p



hsa-miR-199b-5p
hsa-miR-410
hsa-miR-27a-5p



hsa-miR-340-5p
hsa-miR-429
hsa-miR-27b-5p



hsa-miR-34c-5p
hsa-miR-497-5p
hsa-miR-29b-1-5p



hsa-miR-423-3p
hsa-miR-513a-3p
hsa-miR-302a-5p



hsa-miR-489
hsa-miR-542-3p
hsa-miR-3065-5p



hsa-miR-520f
hsa-miR-653
hsa-miR-30d-3p



hsa-miR-520g
hsa-miR-122-3p
hsa-miR-34a-3p



hsa-miR-605
hsa-miR-101-5p
hsa-miR-371a-3p



hsa-miR-668
hsa-miR-1178-3p
hsa-miR-373-5p



hsa-let-7a-5p
hsa-miR-191-5p
hsa-miR-374a-3p



hsa-let-7f-5p
hsa-miR-214-5p
hsa-miR-376a-5p



hsa-miR-10a-3p
hsa-miR-302d-5p
hsa-miR-378a-5p



hsa-miR-135b-5p
hsa-miR-572
hsa-miR-424-3p



hsa-miR-144-3p
hsa-miR-574-3p
hsa-miR-451a



hsa-miR-181d
hsa-miR-26a-2-3p
hsa-miR-452-3p



hsa-miR-200b-3p
hsa-miR-611
hsa-miR-487b



hsa-miR-200c-3p
hsa-let-7f-1-3p
hsa-miR-493-5p



hsa-miR-218-5p
hsa-let-7i-3p
hsa-miR-500a-3p



hsa-miR-23b-3p
hsa-miR-100-5p
hsa-miR-502-3p



hsa-miR-25-3p
hsa-miR-106b-5p
hsa-miR-516b-3p



hsa-miR-29b-3p
hsa-miR-132-5p
hsa-miR-518e-3p



hsa-miR-383
hsa-miR-135b-3p
hsa-miR-518f-3p



hsa-miR-202-3p
hsa-miR-136-3p
hsa-miR-519a-5p



hsa-miR-381-3p
hsa-miR-150-3p
hsa-miR-519b-5p



hsa-miR-377-3p
hsa-miR-154-3p
hsa-miR-521



hsa-miR-452-5p
hsa-miR-15a-3p
hsa-miR-523-5p



hsa-miR-501-3p
hsa-miR-15b-3p
hsa-miR-545-5p



hsa-miR-514a-3p
hsa-miR-16-1-3p
hsa-miR-585



hsa-miR-654-3p
hsa-miR-181a-2-3p
hsa-miR-7-2-3p



hsa-let-7b-3p
hsa-miR-181c-3p
hsa-miR-93-3p



hsa-miR-125a-3p
hsa-miR-186-3p
hsa-miR-96-3p



hsa-miR-133b
hsa-miR-195-3p
hsa-miR-99b-3p



hsa-miR-192-5p
hsa-miR-20b-3p



hsa-miR-199a-3p
hsa-miR-223-5p










c) A Multiple microRNAs-One mRNA Paradigm.

A. One exemplary multiple miRNAs-one mRNA paradigm involves UCP1. In adipocytes the key thermogenic regulator ultimately is UCP1 (also named thermogenin) and, thus, all thermogenic regulators must ultimately impact UCP1 activity. UCP1 is a mitochondrial transporter protein that creates proton leaks across the inner mitochondrial membrane, thus uncoupling oxidative phosphorylation from ATP synthesis. As a result, energy is dissipated in the form of heat (adaptive thermogenesis) (see FIG. 5) Lowell et al., Nature (2000); Friedman et al., Bioinformatics (2010); Hsu et al., Nucleic acids research (2011); Rieger et al., Frontiers in Genetics (2011)).
UCP1 biosynthesis is mainly controlled at the transcription level. FIG. 6 depicts the transcriptional control of UCP1 by other exemplary thermogenic regulators. The promoter's region of the UCP1 gene contains many distinct regulatory sites, allowing a wide range of proteins to influence its transcription, both positively (see FIG. 7A) and negatively (see FIG. 7B).
Mendelian randomization is a method of using measured variation in genes of known function to examine the causal effect of a modifiable exposure on disease in non-experimental studies. Mendelian randomization can be thought of as a “natural” Randomized Clinical Trial. Genetic polymorphism of the UCP1 gene, such as the −3826 A/G single nucleotide polymorphism in the promoter in exon 2 of UCP1, has been reported to be associated with reduced mRNA expression and obesity. Healthy children with the G/G genotype had a lower capacity for thermogenesis in response to a high-fat meal and acute cold exposure. The same −3826 A/G UCP1 genetic polymorphism diminishes resting energy expenditure and thermoregulatory sympathetic nervous system activity in young females. In a study of 367 Korean women, the G allele of −3826A>G and the C allele of −412A>C were significantly associated with larger areas of abdominal subcutaneous fat in a dominant model (p<0.001 and p<0.0004, respectively); combining them together (ht2[GC]) enhanced this significance (p<0.00005). A study of 100 severe obese adults (BMI>40 kg/m2) and 100 normal-weight control subjects (BMI range=19-24.9 kg/m2) identified 7 variations in the promoter region, 4 in the intronic region and 4 in the exonic region of the UCP1 gene. These variations could contribute to the development of obesity, particularly, g.−451C>T, g.940G>A, and g.IVS4-208T>G could represent “thrifty” factors that promote energy storage. Finally, two polymorphisms (A-3826G and C-3740A), located in the upstream promoter region of the UCP1 gene affect gene expression and are correlated with human longevity.
All aforementioned information supports targeting UCP1 expression and activity as a meaningful way to alter adaptive thermogenesis and consequently treat human obesity. Many strategies could be implemented to achieve this goal, however, the one employed in the methods of the invention uses miRNA agents to modulate simultaneously several elements within the thermogenic pathways to increase UCP1 synthesis and activity. Both direct and indirect interactions between miRNAs and the UCP1 gene are considered. Direct interaction means the direct binding of miRNAs to the various regions of the UCP1 gene, resulting in alterations of the transcription, translation, stability and/or degradation of the UCP1 mRNA. Indirect interaction means that miRNAs alter the transcription, translation, stability and/or degradation of thermogenic mRNAs, whose expressed proteins alter the transcription of the UCP1 gene. Furthermore, indirect interaction means that miRNAs alter the transcription, translation, stability and/or degradation of other miRNAs that modify the transcription of the UCP1 gene.
The promoter region of the human UCP1 gene (gi|237858805|ref|NG—012139.1| Homo sapiens uncoupling protein 1 (mitochondrial, proton carrier) (UCP1), RefSeqGene on chromosome 4) is particularly rich is regulatory element motifs:








UCP1 Gene Regulatory Elements:



1. Brown Fat Response Element 1 (BRE1) Motif:


[SEQ ID NO: 1]



CCTCTCTGCTTCTTCT



One


Length: 16, Interval: 1,129 -> 1,144, Mismatches: 0.


 


2. Brown Fat Response Element 2 (BRE2) Motif:


[SEQ ID NO: 2]



CTCCTTGGAA



One


Length: 10, Interval: 1,269 -> 1,278, Mismatches: 0.


 


3. CRE2 Motif:


ATTCTTTA


Four


Length: 8, Intervals: 1,121 -> 1,128, 3,631 -> 3,638, 10,982 -> 10,989,


15,881 -> 15,888, Mismatches: 0.


 


4. CREB Motif:


ACGTCA


Five


Length: 6, Intervals: 1,082 -> 1,087, 1,345 -> 1,350, 1,348 -> 1,343, 11,439 -> 11,434,


13,831 -> 13,836, Mismatches: 0.


 


5. DR1 Motif:


[SEQ ID NO: 3]



TTGCCCTTGCTCA



One


Length: 13, Interval: 1,099 -> 1,111, Mismatches: 0.


 


6. DR4 Motif:


[SEQ ID NO: 4]



ACGTCATAAAGGGTCA



One


Length: 16, Interval: 1,082 -> 1,097, Mismatches: 0.


 


7. DR4 Type RARE Motif:


[SEQ ID NO: 5]



RGKTCANNNNRGKTCA



One


Length: 16, Interval: 1,316 -> 1,301, Mismatches: 0.


 


8. ERE Motif:


[SEQ ID NO: 6]



GCTCATACTGACCT



One


Length: 14, Interval: 1,107 -> 1,120, Mismatches: 0.


 


9. PRE Motif:


[SEQ ID NO: 7]



GTTAATGTGTTCT



One


Length: 13, Interval: 1,009 -> 1,021, Mismatches: 0.


 


10. RARE Motif:


[SEQ ID NO: 8]



TGACCACAGTTTGATCA



One


Length: 17, Interval: 983 -> 999, Mismatches: 0.


 


11. RXR Motif:


AGGTCA


Twelve


Length: 6, Interval: 1,120 -> 1,115, 1,316 -> 1,311, 3,517 -> 3,522, 3,560 -> 3,555, 3,813 ->


3,808, 5,318 -> 5,313, 6,233 -> 6,238, 6,831 -> 6,836, 8,122 -> 8,127, 9,966 -> 9,971,


11,339 -> 11,334, 11,412 -> 11,407, Mismatches: 0.


 


12. GC Box 1 Motif:


CGCCC


Seven


Length: 5, Interval: 4,593 -> 4,589, 4,615 -> 4,619, 4,615 -> 4,619, 4,747 -> 4,751, 4,765 ->


4,769, 5,914 -> 5,910, 13,715 -> 13,711, Mismatches: 0.


 


13. GC Box 2 Motif:


GCGGG


Nine


Length: 5, Interval: 4,463 -> 4,459, 4,585 -> 4,589, 4,593 -> 4,597, 4,639 -> 4,643, 4,883 ->


4,887, 5,176 -> 5,172, 5,929 -> 5,933, 5,940 -> 5,944, 14,994 -> 14,990, Mismatches: 0.


 


14. GT Box 1 Motif:


CACCC


Twenty Five


Length: 5, Interval: 194 -> 190, 452 -> 448, 1,184 -> 1,188, 1,803 -> 1,807, 2,428 -> 2,424,


3,037 -> 3,041, 3,330 -> 3,334, 4,137 -> 4,141, 4,566 -> 4,562, 4,599 -> 4,595, 4,869 -> 4,865,


5,104 -> 5,108, 5,461 -> 5,457, 6,237 -> 6,241, 6,293 -> 6,289, 8,096 -> 8,092, 8,198 -> 8,194,


9,649 -> 9,645, 9,912 -> 9,908, 12,962 -> 12,958, 13,136 -> 13,132, 13,723 -> 13,719, 14,404 ->


14,400, 14,960 -> 14,964, 15,576 -> 15,572, Mismatches: 0.


 


15. GT Box 2 Motif:


GTGGG


Twenty


Length: 5, Interval: 25 -> 21, 1,805 -> 1,801, 1,809 -> 1,805, 2,119 -> 2,123, 3,854 -> 3,850,


4,310 -> 4,314, 4,339 -> 4,343, 4,765 -> 4,761, 4,867 -> 4,871, 6,291 -> 6,295, 7,554 -> 7,558,


8,280 -> 8,284, 8,681 -> 8,685, 9,615 -> 9,619, 9,689 -> 9,693, 9,906 -> 9,910, 10,363 ->


10,359, 13,074 -> 13,070, 13,640 -> 13,644, 13,941 -> 13,945, Mismatches: 0.


 


16. CpG Methylation Island Motif: CG


Three Hundred and Sixty Six, including many between positions 4,519 to 5,258 and 5,639 to


6,694.






FIG. 8A depicts the location of these various regulatory elements in reference to the UCP1 transcription start site at nucleotide position 5,001 of the 15,910 base pair human UCP-1 gene (FASTA accession number: >gi|237858805|ref|NG—012139.1| Homo sapiens uncoupling protein 1 (mitochondrial, proton carrier) (UCP1), RefSeqGene on chromosome 4; NCBI Reference Sequence: NG—012139.1).
Direct or indirect activation or repression of these regulatory elements by miRNAs will result in alterations of UCP1 gene expression and activity. Under normal conditions, the UCP1 gene expression and activity are repressed by a rich network of regulatory elements, in order to avoid energy wasting. Under stress, such as exposure to a cold environment, the expression of the UCP1 gene is upregulated, via various activators and repressors which are under the control of several miRNAs.
An initial survey of miRNAs targeting the human UCP1 3′UTR with several programs, including microRNA.org, was negative. However, other programs, including MicroCosm Targets, using the UCP1 Ensembl 1,462 base pair transcript ENST00000262999 as a target revealed binding sites for 27 miRNAs at 28 locations in UCP1 3′UTR as shown in Table 6.







TABLE 6







Binding sites for miRNAs in the 3′UTR of UCP1 (NCBI Reference Sequence.


NG_012139.1) determined using microCosm Targets.














SEQ







ID





Name
Sequence
NO
Minimum
Maximum
Length















hsa-miR-21
AATGTAATGCAGATAAGCTA
9
14143
14162
20


 


hsa-miR-219-2-3p
ACATGTTTTAATTACAATTC
10
14217
14236
20


 


hsa-miR-22
GATTGGCAGCTT
11
14857
14868
12


 


hsa-miR-222a
GATTTTTAATGTTTAGAGTCCAG
12
14500
14522
23


 


hsa-miR-290-3p
TTTAGAGCTGGAGGGTACTT
13
14621
14640
20


 


hsa-miR-292-3p
TTTAGAGCTGGAGGGTACTT
14
14621
14640
20


 


hsa-miR-292-5p
GACAGAGGAACAGTTTGAG
15
14648
14666
19


 


hsa-miR-325
ATTTTGGCAGGATTGCTACTAG
16
14568
14589
22


 


hsa-miR-331-5p
TTTTGAGATCTATACCTGG
17
14383
14401
19


 


hsa-miR-362-5p
ATTTTAAGCTAAATCCAAGGATT
18
14838
14860
23


 


hsa-miR-367
TGACCATTTCTGGAGTGCAATT
19
14170
14191
22


 


hsa-miR-371-5p
ACAGTTTGAT
20
988
997
10


 


hsa-miR-371-5p
ACAGTTTGAG
21
14657
14666
10


 


hsa-miR-377
CTGGAGTGCAATTGTGTGA
22
14179
14197
19


 


hsa-miR-378
TTTTAATGTTTAGAGTCCAG
23
14503
14522
20


 


hsa-miR-382
TGATGACATCTCTAACAACTTC
24
14526
14547
22


 


hsa-miR-460
AGAAACTGAGTGAAATGCAG
25
14250
14269
20


 


hsa-miR-508-5p
TGACCATTTCTGGAGTG
26
14170
14186
17


 


hsa-miR-543
TACTCTGAATGTT
27
14478
14490
13


 


hsa-miR-549
TTAACCACAGTTGTCA
28
14321
14336
16


 


hsa-miR-643
CAAGTTCACTAGAATACAAG
29
14412
14431
20


 


hsa-miR-654-3p
AAGGTTACAGGCTGCCAGACAT
30
14880
14901
22


 


hsa-miR-664
GTGTGAATGAATG
31
14192
14204
13


 


hsa-miR-871
TAGGCATGAACCTACTCTGAATG
32
14466
14488
23


 


hsa-miR-883a-3p
AAACTGAGTGAAATGCAGTT
33
14252
14271
20


 


hsa-miR-883b-3p
AAACTGAGTGAAATGCAGTT
34
14252
14271
20


 


hsa-miR-888-3p
TTTATTAACCACAGTTGTCAGTT
35
14317
14339
23


 


hsa-miR-92b
GAGTGCAAT

14182
14190
9









Other programs, such as miRWalk, miRGen, miRGator-miRanda, and DIANA microT, using the UCP1 Ensembl 1,462 base pair transcript (ENST00000262999), the UCP1 Ensembl 9,371 base pair gene sequence (ENSG00000109424) or the 15,910 base pair UCP1 sequence (NCBI Reference Sequence: NG—012139.1) as targets, revealed binding sites for a total of 50 miRNAs at 69 locations in UCP1 3′UTR as shown in Table 7.







TABLE 7







Binding sites for miRNAs in the 3′UTR of UCP1 (NCBI Reference Sequence.


NG_012139.1) according to several programs.














Name
Sequence
SEQ ID NO
Minimum
Maximum
Length
















1
hsa-miR-1179
AAGTATCCTTT
36
15346
15356
11


 


2
hsa-miR-1302
ATGGGACACA
37
15021
15030
10


 


3
hsa-miR-130b
TTATTTTCCCT
38
15161
15171
11


 


4
hsa-miR-146a
TGACAACTGT
39
14327
14336
10


 



hsa-miR-146a
AGGGAACTGA
40
15231
15240
10


 



hsa-miR-146a
TGTGAACTGG
41
15679
15688
10


 


5
hsa-miR-181c
AACCATAGT

15304
15312
9


 


6
hsa-miR-19b-2
ACTTTTGCGG
42
14991
15000
10


 


7
hsa-miR-203
TTAAATGTT

15584
15592
9


 


8
hsa-miR-204-5p
TTCCTTTATC
43
14006
14015
10


 



hsa-miR-204-5p
TTCCTCTGTC
44
14648
14657
10


 


9
hsa-miR-21-5p
TAGCTTATCT
45
14153
14162
10


 


10
hsa-miR-211-5p
TTCCCTATCTC
46
14779
14789
11


 


11
hsa-miR-214
CAGCAAGCA
47
15052
15060
9


 


12
hsa-miR-22-3p
AAGCTGCCAA
48
14859
14868
10


 



hsa-miR-22-5p
AGTTCTTCACA
49
14203
14213
11


 


13
hsa-miR-26a-2-3p
CATTTTCTTG
50
13918
13927
10


 



hsa-miR-26a-2-3p
CCAATCCTTG
51
14853
14862
10


 



hsa-miR-26a-2-3p
CCTTTTCATG
52
15616
15625
10


 


14
hsa-miR-30b
GTAACCTTCC
53
14878
14887
10


 


15
hsa-miR-325
CAGAGTAGGT
54
14475
14484
10


 



hsa-miR-325
CCTTGTAGGC
55
15378
15387
10


 


16
hsa-miR-328
CTGTTCCTCT
56
14651
14660
10


 


17
hsa-miR-362-5p
ATCCTTGGAT
57
14850
14859
10


 


18
hsa-miR-367-3p
AATTGCACTC
58
14182
14191
10


 


19
hsa-miR-371a-3p
AAGTGCCTGC
59
15435
15444
10


 



hsa-miR-371a-5p
TCTCAAACTG
60
14658
14667
10


 


20
hsa-miR-378a-3p
ACTGGCCTTG
61
15816
15825
10


 


21
hsa-miR-382-3p
ATTCATTCAC
62
14194
14203
10


 


22
hsa-miR-382-5p
GAAGTTGTTAGAGAT
63
14533
14547
15


 


23
hsa-miR-383
AGATTAGAA
64
14545
14553
9


 


24
hsa-miR-421
ATTAACTGAC
65
14333
14342
10


 



hsa-miR-421
CTCAAAAGAC
66
14380
14389
10


 


25
hsa-miR-422a
ACTGGCCTT

15817
15825
9


 


26
hsa-miR-431
TGTCTGGCA

14892
14900
9


 


27
hsa-miR-452
TTATCTGC

14151
14158
8


 



hsa-miR-452
TCTTCTGC

14773
14780
8


 



hsa-miR-452
ACATCTGC

15009
15016
8


 


28
hsa-miR-455-3p
CAGTCCAT

13893
13900
8


 



hsa-miR-455-5p
TGTGTGCCTT
67
15641
15650
10


 


29
hsa-miR-491-5p
AATGGGGAAG
68
14975
14984
10


 


30
hsa-miR-501-3p
ATGCATCAGG
69
15547
15556
10


 


31
hsa-miR-504
AGACCCTGT

15325
15333
9


 




TATTCTAGTGAACTTG
70





 


32
hsa-miR-508-5p
ACTCTTA

14405
14427
23


 


33
hsa-miR-512-5p
CACTCAG

14255
14261
7


 


34
hsa-miR-514a-3p
TTGACTCTT

14406
14414
9


 


35
hsa-miR-515-3p
GACTGCCTT

15539
15547
9


 



hsa-miR-515-3p
GTGTGCCTT

15641
15649
9


 


36
hsa-miR-517a-3p
ATGGTGCATT
71
15650
15659
10


 


37
hsa-miR-545
CAGCAAGCACT
72
15050
15060
11


 


38
hsa-miR-549
TGACAACTGT
73
14327
14336
10


 


39
hsa-miR-552
CACAGGTGA

15130
15138
9


 


40
hsa-miR-616-5p
ACTCTAAAC

14510
14518
9


 


41
hsa-miR-620
ATGAATATAG
74
14560
14569
10


 


42
hsa-miR-643
ACTGGTATGT
75
13933
13942
10


 



hsa-miR-643
TCTTGTATTC
76
14423
14432
10


 



hsa-miR-643
CCTTGTAGGC
77
15378
15387
10


 



hsa-miR-643
ACATGCATGC
78
15553
15562
10


 


43
hsa-miR-651
TTAAAATAAG
79
13988
13997
10


 



hsa-miR-651
TTAGGTTAAA
80
13993
14002
10


 



hsa-miR-651
TCATGATAAG
81
15700
15709
10


 


44
hsa-miR-654-3p
TATCTCTTCT
82
14775
14784
10


 



hsa-miR-654-3p
TATGTATACT
83
15493
15502
10


 


45
hsa-miR-655
GTAATACAT

15593
15601
9


 


46
hsa-miR-767-3p
CCTGCTCAT

14871
14879
9


 


47
hsa-miR-888-3p
GACTGACTCC
84
15772
15781
10


 


48
hsa-miR-92b-3p
ATTGCACTCC
85
14181
14190
10


 


49
hsa-miR-941
CACCCAGGT

14396
14404
9


 


50
hsa-miR-99a-3p
AAGCTGGCTC
86
15117
15126
10









Alignment of the sequence of the human UCP1 gene with several miRNA sequences yielded matches in the 5′UTR, the promoter region and the coding regions of the UCP1 gene. Interrogation of the publicly available Internet tools predicting miRNAs targeting the various regions of the UCP1 gene elicited several hits. Surprisingly, the overlap between these prediction tools was zero, as shown in FIG. 3.
Nevertheless, miRNA databases were screened using the alignment program Geneious. A total of 191 human microRNAs were found which have complementary 450 binding sites in the UCP1 gene sequence (Table 8). The length of the matches goes from 7 bases to 12 bases (e.g. hsa-miR-24-2-5p and hsa-miR-192-5p). The number of hits per miRNA varies from 1 to several (e.g. 9 for hsa-miR-19b2 (an abundant adipocyte miRNA), 14 for hsa-miR-26a-2-3p, 11 for hsa-miR-181c, and 12 for hsa-miR-620).







TABLE 8







miRNAs with predicted binding sites in the UCP1 gene sequence (NCBI


Reference Sequence: NG_012139.1).















SEQ ID






miRNA
Sequence
NO
Minimum
Maximum
Length
Direction
















hsa-let-7c
TAGAGTTTC

5918
5926
9
reverse


 


hsa-let-7e
GGAGGTAGG

13283
13291
9
reverse


 


hsa-let-7e
TGAAGTAGG

7612
7620
9
reverse


 


hsa-let-7e
AGAGGTAGG

3306
3314
9
reverse


 


hsa-let-7i-3p
CTGTGCAAG

3588
3596
9
reverse


 


hsa-miR-17
CAAAGTGCT

12200
12208
9
reverse


 


hsa-miR-17
CAAAGTGCT

9931
9939
9
reverse


 


hsa-miR-17
CAAAGTGCT

218
226
9
reverse


 


hsa-miR-19a
TGTGCAAAT

3916
3924
9
reverse


 


hsa-miR-19a
TGTGCAAAT

834
842
9
reverse


 


hsa-miR-19b-2
ACTTTTGCGG
87
14991
15000
10
reverse


 


hsa-miR-19b-2
AGTTTTACAA
88
11998
12007
10
reverse


 


hsa-miR-19b-2
AGTTTTGTAT
89
10023
10032
10
reverse


 


hsa-miR-19b-2
AGTCTTGAAG
90
9399
9408
10
reverse


 


hsa-miR-19b-2
AGGTTTGTAG
91
7758
7767
10
reverse


 


hsa-miR-19b-2
AGTATTGAAG
92
7159
7168
10
reverse


 


hsa-miR-19b-2
AGGCTTGCAG
93
3546
3555
10
reverse


 


hsa-miR-19b-2
AATTTGGCAG
94
529
538
10
reverse


 


hsa-miR-19b-2
AGTTTTGGAA
95
312
321
10
reverse


 


hsa-miR-20b
CAAAGTGCT

12200
12208
9
reverse


 


hsa-miR-20b
CAAAGTGCT

9931
9939
9
reverse


 


hsa-miR-20b
CAAAGTGCT

218
226
9
reverse


 


hsa-miR-21-5p
TAGCTTATCT
96
14153
14162
10
reverse


 


hsa-miR-22-3p
AAGCTGCCAA
97
14859
14868
10
reverse


 


hsa-miR-22-3p
AAGCTTCCAG
98
1482
1491
10
reverse


 


hsa-miR-22-5p
AGTTCTTCACA
99
14203
14213
11
reverse


 


hsa-miR-22-5p
AATTCTTCAGG
100
8032
8042
11
reverse


 


hsa-miR-22-5p
GGTTCTTCAGC
101
5389
5399
11
reverse


 


hsa-miR-24-2-5p
TGCCTACTGGCC
102
8651
8662
12
reverse


 


hsa-miR-25-3p
CATTGCAC

11565
11572
8
reverse


 


hsa-miR-25-5p
AGGCGGAG

5963
5970
8
reverse


 


hsa-miR-26a-2-3p
CCTTTTCATG
103
15616
15625
10
reverse


 


hsa-miR-26a-2-3p
CCAATCCTTG
104
14853
14862
10
reverse


 


hsa-miR-26a-2-3p
CATTTTCTTG
105
13918
13927
10
reverse


 


hsa-miR-26a-2-3p
CCTACTCTTC
106
13505
13514
10
reverse


 


hsa-miR-26a-2-3p
ACGATTCTTG
107
13192
13201
10
reverse


 


hsa-miR-26a-2-3p
TCTATTCTTT
108
12883
12892
10
reverse


 


hsa-miR-26a-2-3p
CATATTTTTG
109
10197
10206
10
reverse


 


hsa-miR-26a-2-3p
GCTAGTCTTG
110
9978
9987
10
reverse


 


hsa-miR-26a-2-3p
CATATTTTTG
111
9890
9899
10
reverse


 


hsa-miR-26a-2-3p
CCTTTTCTTT
112
6631
6640
10
reverse


 


hsa-miR-26a-2-3p
CCCATTCTCG
113
4709
4718
10
reverse


 


hsa-miR-26a-2-3p
TTTATTCTTG
114
3893
3902
10
reverse


 


hsa-miR-26a-2-3p
CCTTTACTTG
115
1885
1894
10
reverse


 


hsa-miR-26a-2-3p
GCGATTCTTG
116
376
385
10
reverse


 


hsa-miR-27-5p
AGAGCTTAGG
117
2949
2958
10
reverse


 


hsa-miR-30b
GTAACCTTCC
118
14878
14887
10
reverse


 


hsa-miR-30b
GTAACCATCA
119
12991
13000
10
reverse


 


hsa-miR-30b
GTAATCATAC
120
12831
12840
10
reverse


 


hsa-miR-30b
GTCAACATCA
121
11401
11410
10
reverse


 


hsa-miR-30b
GTAAACATAA
122
9365
9374
10
reverse


 


hsa-miR-30b
GTACTCATCC
123
9016
9025
10
reverse


 


hsa-miR-30b
CTATACATCC
124
8586
8595
10
reverse


 


hsa-miR-30b
CTAAACATCT
125
7495
7504
10
reverse


 


hsa-miR-31
GGCTATGCC

7712
7720
9
reverse


 


hsa-miR-32
ATTGCACA

11564
11571
8
reverse


 


hsa-miR-92b
ATTGCACTCC
126
14181
14190
10
reverse


 


hsa-miR-92b
ATTGCACTAG
127
11282
11291
10
reverse


 


hsa-miR-93
CAAAGTGCTG
128
12199
12208
10
reverse


 


hsa-miR-93
CAAAGTGCTG
129
217
226
10
reverse


 


hsa-miR-93-3p
ACTCCTGGGCT
130
12356
12366
11
reverse


 


hsa-miR-93-3p
ACTGATAAGCT
131
11055
11065
11
reverse


 


hsa-miR-93-3p
ACTCCTGACCT
132
9966
9976
11
reverse


 


hsa-miR-96-3p
AATCATGTGCC
133
8659
8669
11
reverse


 


hsa-miR-99a-3p
AAGCTGGCTC
134
15117
15126
10
reverse


 


hsa-miR-99a-3p
AAACTCTTTC
135
13344
13353
10
reverse


 


hsa-miR-99a-3p
AATCTTGTTC
136
11952
11961
10
reverse


 


hsa-miR-99a-3p
AAGCTCCTTT
137
11050
11059
10
reverse


 


hsa-miR-99a-3p
AAGCTCCTTT
138
8099
8108
10
reverse


 


hsa-miR-99a-3p
AAGCTCTGTC
139
7523
7532
10
reverse


 


hsa-miR-99b-3p
CAACCTCGAG
140
13666
13675
10
reverse


 


hsa-miR-99b-3p
CGAGCTCCTG
141
13660
13669
10
reverse


 


hsa-miR-99b-3p
GAAGCTTGTG
142
6436
6445
10
reverse


 


hsa-miR-99b-3p
CAAACTCCTG
143
257
266
10
reverse


 


hsa-miR-100
TCCAGTAGAT
144
11866
11875
10
reverse


 


hsa-miR-100
ACGCGCAGAT
145
5634
5643
10
reverse


 


hsa-miR-106b-5p
CAAAGTGCTG
146
12199
12208
10
reverse


 


hsa-miR-106b-5p
CAAAGTGCTG
147
217
226
10
reverse


 


hsa-miR-126-3P
TCATACAGT

12828
12836
9
reverse


 


hsa-miR-126-3P
TTGTACTGT

11542
11550
9
reverse


 


hsa-miR-126-3P
TGGTCCCGT

7922
7930
9
reverse


 


hsa-miR-126-3P
TCATACAGT

932
940
9
reverse


 


hsa-miR-130b
TTATTTTCCCT
148
15161
15171
11
reverse


 


hsa-miR-130b
CTCTTTTCAGT
149
9670
9680
11
reverse


 


hsa-miR-130b
CTCTCTTCACT
150
8977
8987
11
reverse


 


hsa-miR-130b
CTCTTTTTCCC
151
8444
8454
11
reverse


 


hsa-miR-130b
CTTTTTCCCCT
152
6624
6634
11
reverse


 


hsa-miR-130b
CTATTTTCCGT
153
5742
5752
11
reverse


 


hsa-miR-130b
TTCCTTTCCCT
154
5007
5017
11
reverse


 


hsa-miR-130b
CTCTTTGCCCC
155
1845
1855
11
reverse


 


hsa-miR-130b
CTCCTTTCCTT
156
1033
1043
11
reverse


 


hsa-miR-133a-1
TTTGGTGCCC
157
7393
7402
10
reverse


 


hsa-miR-140-3p
TACCACAG

5893
5900
8
reverse


 


hsa-miR-141
TAACACTG

5852
5859
8
reverse


 


hsa-miR-143
GGTGCAGTG
158
4132
4140
9
reverse


 


hsa-miR-143-3p
TGAGATGAGG
159
13727
13736
10
reverse


 


hsa-miR-143-3p
TGAGATGGAG
160
10172
10181
10
reverse


 


hsa-miR-143-3p
TTAGATGAAG
161
9572
9581
10
reverse


 


hsa-miR-144-3p
TACAGTATT

12825
12833
9
reverse


 


hsa-miR-144-3p
TACAATATA

8859
8867
9
reverse


 


hsa-miR-144-3p
GACAGTATA

1491
1499
9
reverse


 


hsa-miR-146a
CCTCTGAAA

3499
3507
9
reverse


 


hsa-miR-146a
TGTGAACTGG
162
15679
15688
10
reverse


 


hsa-miR-146a
AGGGAACTGA
163
15231
15240
10
reverse


 


hsa-miR-146a
TGACAACTGT
164
14327
14336
10
reverse


 


hsa-miR-146a
TAAGAACTAA
165
8935
8944
10
reverse


 


hsa-miR-146a
TTAGAACAGA
166
7908
7917
10
reverse


 


hsa-miR-146a
TGAGAAGTGC
167
6926
6935
10
reverse


 


hsa-miR-146a
TGAAAACTTA
168
3883
3892
10
reverse


 


hsa-miR-146a
ACAGAACTGA
169
2259
2268
10
reverse


 


hsa-miR-146a
TGAGACCAGA
170
2235
2244
10
reverse


 


hsa-miR-146a
TGAGAAATAA
171
1614
1623
10
reverse


 


hsa-miR-147
TGTGTGGATAA
172
7223
7233
11
reverse


 


hsa-miR-147
TTTGTGCAAAT
173
3916
3926
11
reverse


 


hsa-miR-154
AATCATACA

12830
12838
9
reverse


 


hsa-miR-154
AATCATACA

934
942
9
reverse


 


hsa-miR-181c
AACCATAGT

15304
15312
9
reverse


 


hsa-miR-181c
AACCAAAGA

13244
13252
9
reverse


 


hsa-miR-181c
AACCATCAC

12990
12998
9
reverse


 


hsa-miR-181c
ATCCAGCGA

11466
11474
9
reverse


 


hsa-miR-181c
AAACATCTA

7494
7502
9
reverse


 


hsa-miR-181c
AAAAATCGA

6201
6209
9
reverse


 


hsa-miR-181c
AACCCCCGA

5540
5548
9
reverse


 


hsa-miR-181c
AACCCTCTA

3614
3622
9
reverse


 


hsa-miR-181c
AGCCAGCGA

3471
3479
9
reverse


 


hsa-miR-181c
AACCATAGG

2801
2809
9
reverse


 


hsa-miR-181c
AACCATCAC

194
202
9
reverse


 


hsa-miR-185
TGGAGAGAA

2979
2987
9
reverse


 


hsa-miR-192-5p
CTAACATATGAA
174
114
125
12
reverse


 


hsa-miR-194-1
TGTAACAGCA
175
1895
1904
10
reverse


 


hsa-miR-196a
AGGTAGTTT

12139
12147
9
reverse


 


hsa-miR-199a-5p
CCCTGTGTTC
176
5753
5762
10
reverse


 


hsa-miR-200a
TAACACTG

5852
5859
8
reverse


 


hsa-miR-200b
TAATAATGCC
177
11184
11193
10
reverse


 


hsa-miR-200b
GAATACTGCC
178
10340
10349
10
reverse


 


hsa-miR-200c-3p
TAATACTGT

12466
12474
9
reverse


 


hsa-miR-200c-3p
TAATAATGC

11185
11193
9
reverse


 


hsa-miR-200c-3p
GAATACTGC

10341
10349
9
reverse


 


hsa-miR-200c-3p
TAATACAGC

7594
7602
9
reverse


 


hsa-miR-203
TTAAATGTT

15584
15592
9
reverse


 


hsa-miR-203
TGAAATTTT

9782
9790
9
reverse


 


hsa-miR-203
TGAAAGGTT

4495
4503
9
reverse


 


hsa-miR-204-5p
TTCCTCTGTC
179
14648
14657
10
reverse


 


hsa-miR-204-5p
TTCCTTTATC
180
14006
14015
10
reverse


 


hsa-miR-205
TCCTTCATT

10659
10667
9
reverse


 


hsa-miR-208b
ATAAGAAGA

9493
9501
9
reverse


 


hsa-miR-208b
ATAAGAAGA

1770
1778
9
reverse


 


hsa-miR-211-5p
TTCCCTATCTC
181
14779
14789
11
reverse


 


hsa-miR-211-5p
TCCCCTCTGTC
182
5238
5248
11
reverse


 


hsa-miR-211-5p
TTCCCTTGCTC
183
5002
5012
11
reverse


 


hsa-miR-211-5p
TTCCCATTCTC
184
4710
4720
11
reverse


 


hsa-miR-214
CAGCAAGCA

15052
15060
9
reverse


 


hsa-miR-214
CAGAAGGCA

6918
6926
9
reverse


 


hsa-miR-214
CCGCAGGCA

5935
5943
9
reverse


 


hsa-miR-214
CACCAGGCA

2087
2095
9
reverse


 


hsa-miR-218
TGTGCTTGA

10385
10393
9
reverse


 


hsa-miR-302c
TTTAACATG

2932
2940
9
reverse


 


hsa-miR-324-5p
CGCGTCCCCT
185
4876
4885
10
reverse


 


hsa-miR-325
CCTTGTAGGC
186
15378
15387
10
reverse


 


hsa-miR-325
CAGAGTAGGT
187
14475
14484
10
reverse


 


hsa-miR-325
CCAAGTAGCT
188
10066
10075
10
reverse


 


hsa-miR-325
CCAAGTAGCT
189
354
363
10
reverse


 


hsa-miR-328
CTGTTCCTCT
190
14651
14660
10
reverse


 


hsa-miR-328
CTGGCTCCCT
191
8215
8224
10
reverse


 


hsa-miR-328
CTGGCCCTTC
192
8062
8071
10
reverse


 


hsa-miR-328
CTGGCACTCA
193
6653
6662
10
reverse


 


hsa-miR-328
CTGGCTTTCT
194
6496
6505
10
reverse


 


hsa-miR-328
CTGCCCCTCC
195
6048
6057
10
reverse


 


hsa-miR-328
CTGGGCCGCT
196
4804
4813
10
reverse


 


hsa-miR-328
CTGGAGCTCT
197
4477
4486
10
reverse


 


hsa-miR-328
CTGACCCTTT
198
1089
1098
10
reverse


 


hsa-miR-330
CAAAGCACAC
199
13845
13854
10
reverse


 


hsa-miR-330
CAAAGCACAC
199
11657
11666
10
reverse


 


hsa-miR-331-5p
CTAGGTGTGG
200
7719
7728
10
reverse


 


hsa-miR-361-3p
CCCCCAGG

5112
5119
8
reverse


 


hsa-miR-362-5p
ATCCTTGGAT
201
14850
14859
10
reverse


 


hsa-miR-367-3p
AATTGCACTC
202
14182
14191
10
reverse


 


hsa-miR-367-3p
AAATGCACTT
203
999
1008
10
reverse


 


hsa-miR-369
AATAATACA

2266
2274
9
reverse


 


hsa-miR-371a-3p
AAGTGCCTGC
204
15435
15444
10
reverse


 


hsa-miR-371a-3p
AAGAGCCGAC
205
11455
11464
10
reverse


 


hsa-miR-371a-3p
ACGTGCCACC
206
10044
10053
10
reverse


 


hsa-miR-371a-3p
AAGTGCCTCT
207
7047
7056
10
reverse


 


hsa-miR-371a-3p
AAGTGCACCC
208
5457
5466
10
reverse


 


hsa-miR-371a-5p
TCTCAAACTG
209
14658
14667
10
reverse


 


hsa-miR-372
AAAGTGCTG

12199
12207
9
reverse


 


hsa-miR-372
AAAGTGCTG

217
225
9
reverse


 


hsa-miR-374a-3p
TCATCAGATT
210
10606
10615
10
reverse


 


hsa-miR-377-3p
AGCACACAAA
211
13842
13851
10
reverse


 


hsa-miR-378a-3p
ACTGGCCTTG
212
15816
15825
10
reverse


 


hsa-miR-378a-3p
ACTGGTCTTG
213
11837
11846
10
reverse


 


hsa-miR-378a-5p
CTCCTGCCTC
214
12216
12225
10
reverse


 


hsa-miR-378a-5p
CTCCTGCCTC
215
10082
10091
10
reverse


 


hsa-miR-378a-5p
CTCCTGTCTC
216
8207
8216
10
reverse


 


hsa-miR-378a-5p
CTCCTAACTC
217
7650
7659
10
reverse


 


hsa-miR-382-3p
ATTCATTCAC
218
14194
14203
10
reverse


 


hsa-miR-383
AGATTAGAA

14545
14553
9
reverse


 


hsa-miR-383
AGATTAGAA

7912
7920
9
reverse


 


hsa-miR-383
AGAACAGAA

5801
5809
9
reverse


 


hsa-miR-412
ACTTCACCT

737
745
9
reverse


 


hsa-miR-421
CTCAAAAGAC
219
14380
14389
10
reverse


 


hsa-miR-421
ATTAACTGAC
220
14333
14342
10
reverse


 


hsa-miR-421
AACATCAGAC
221
11398
11407
10
reverse


 


hsa-miR-421
ATCAACTGAG
222
3427
3436
10
reverse


 


hsa-miR-421
ATCAACAGGT
223
2443
2452
10
reverse


 


hsa-miR-421
ATCAAAAGAT
224
2333
2342
10
reverse


 


hsa-miR-422a
ACTGGCCTT

15817
15825
9
reverse


 


hsa-miR-422a
ACTGGTCTT

11838
11846
9
reverse


 


hsa-miR-422a
ACTGGACGT

5847
5855
9
reverse


 


hsa-miR-425
AGCGGGAAGGT
225
5167
5177
11
reverse


 


hsa-miR-431
TGTCTGGCA

14892
14900
9
reverse


 


hsa-miR-431
TGTCTAGCA

9218
9226
9
reverse


 


hsa-miR-432-5p
TCCTGGAGT

13624
13632
9
reverse


 


hsa-miR-432-5p
TATTGGAGT

10785
10793
9
reverse


 


hsa-miR-432-5p
TCTTAGAGT

9263
9271
9
reverse


 


hsa-miR-432-5p
TCTTAGAGT

6666
6674
9
reverse


 


hsa-miR-432-5p
TCTTGGAGC

2180
2188
9
reverse


 


hsa-miR-452
ACATCTGC

15009
15016
8
reverse


 


hsa-miR-452
TCTTCTGC

14773
14780
8
reverse


 


hsa-miR-452
TTATCTGC

14151
14158
8
reverse


 


hsa-miR-452
TCCTCTGC

13488
13495
8
reverse


 


hsa-miR-452
TCATGTGC

8660
8667
8
reverse


 


hsa-miR-452
TCATCTGG

8221
8228
8
reverse


 


hsa-miR-452
TCATGTGC

7945
7952
8
reverse


 


hsa-miR-452
ACATCTGC

7508
7515
8
reverse


 


hsa-miR-452
CCATCTGC

6787
6794
8
reverse


 


hsa-miR-452
TCATCCGC

5912
5919
8
reverse


 


hsa-miR-452
TCATCTGT

4053
4060
8
reverse


 


hsa-miR-452
TCATCTCC

3667
3674
8
reverse


 


hsa-miR-452
TCCTCTGC

3457
3464
8
reverse


 


hsa-miR-452
TCTTCTGC

2210
2217
8
reverse


 


hsa-miR-455-3p
CAGTCCAT

13893
13900
8
reverse


 


hsa-miR-455-5p
TGTGTGCCTT
226
15641
15650
10
reverse


 


hsa-miR-455-5p
TCTGTGCCTT
227
11203
11212
10
reverse


 


hsa-miR-455-5p
TATGTGCTTT
228
10522
10531
10
reverse


 


hsa-miR-483-3p
CACTCCTC

13536
13543
8
reverse


 


hsa-miR-483-3p
CACTCCTC

10333
10340
8
reverse


 


hsa-miR-483-3p
CACTCCTC

6101
6108
8
reverse


 


hsa-miR-486-5p
TCATGTACT

9835
9843
9
reverse


 


hsa-miR-486-5p
TCCTGTCCT

6526
6534
9
reverse


 


hsa-miR-487a
AATCATACAG
229
12829
12838
10
reverse


 


hsa-miR-487a
AATCATACAG
229
933
942
10
reverse


 


hsa-miR-491-5p
AATGGGGAAG
230
14975
14984
10
reverse


 


hsa-miR-491-5p
AGAGGGGACC
231
12315
12324
10
reverse


 


hsa-miR-491-5p
AGTTGGGCAC
232
11555
11564
10
reverse


 


hsa-miR-491-5p
AGTAGAGAAC
233
6909
6918
10
reverse


 


hsa-miR-491-5p
GGTGAGGAAC
234
6005
6014
10
reverse


 


hsa-miR-491-5p
AGCGGGGCAC
235
4455
4464
10
reverse


 


hsa-miR-491-5p
AGTGGGAAAT
236
3846
3855
10
reverse


 


hsa-miR-496
TTAGTATTA

10948
10956
9
reverse


 


hsa-miR-496
TGAGTATAA

10768
10776
9
reverse


 


hsa-miR-496
TCAGTATTA

9666
9674
9
reverse


 


hsa-miR-501-3p
ATGCATCAGG
237
15547
15556
10
reverse


 


hsa-miR-501-3p
ATCCACCGGG
238
11497
11506
10
reverse


 


hsa-miR-501-3p
AGGCACCAGG
239
2089
2098
10
reverse


 


hsa-miR-504
AGACCCTGT

15325
15333
9
reverse


 


hsa-miR-504
AGCCCCTGG

12898
12906
9
reverse


 


hsa-miR-504
AGTCCCTGG

10591
10599
9
reverse


 


hsa-miR-504
AGACCCGGG

4767
4775
9
reverse


 


hsa-miR-508-3p
TGATTATAGC
240
13565
13574
10
reverse


 


hsa-miR-508-3p
TGAGTGTAGC
241
3231
3240
10
reverse


 


hsa-miR-512-3p
CAGTGCTGTC
242
13211
13220
10
reverse


 


hsa-miR-512-3p
AAGTGCTCTC
243
7688
7697
10
reverse


 


hsa-miR-512-3p
AAGTGCTCTC
243
3184
3193
10
reverse


 


hsa-miR-512-5p
CACTCAG

14255
14261
7
reverse


 


hsa-miR-512-5p
CACTCAG

13591
13597
7
reverse


 


hsa-miR-512-5p
CACTCAG

12291
12297
7
reverse


 


hsa-miR-512-5p
CACTCAG

6652
6658
7
reverse


 


hsa-miR-512-5p
CACTCAG

5067
5073
7
reverse


 


hsa-miR-514a-3p
TTGACTCTT

14406
14414
9
reverse


 


hsa-miR-514a-3p
TTGACAGTT

13870
13878
9
reverse


 


hsa-miR-514a-3p
TTAACACTT

11237
11245
9
reverse


 


hsa-miR-514a-3p
ATGACACTT

10617
10625
9
reverse


 


hsa-miR-515-3p
GTGTGCCTT

15641
15649
9
reverse


 


hsa-miR-515-3p
GACTGCCTT

15539
15547
9
reverse


 


hsa-miR-515-3p
GAGTGACTT

1371
1379
9
reverse


 


hsa-miR-516a-3p
TGCTTCCT

10301
10308
8
reverse


 


hsa-miR-517a-3p
ATGGTGCATT
244
15650
15659
10
reverse


 


hsa-miR-517a-3p
ATCTTGCTTC
245
10303
10312
10
reverse


 


hsa-miR-519b-3p
AAAGTGCAT

13782
13790
9
reverse


 


hsa-miR-519e-3p
AAGTGCCTC

7048
7056
9
reverse


 


hsa-miR-520a-5p
CTCCAGATGG

6274
6283
10
reverse


 


hsa-miR-545
CAGCAAGCACT
246
15050
15060
11
reverse


 


hsa-miR-545
CAGAACACATT
247
11639
11649
11
reverse


 


hsa-miR-545
CTGCAAACACT
248
3450
3460
11
reverse


 


hsa-miR-549
TGACAACTGT
249
14327
14336
10
reverse


 


hsa-miR-551b-3p
GCTACCCAT

2411
2419
9
reverse


 


hsa-miR-552
CACAGGTGA

15130
15138
9
reverse


 


hsa-miR-552
AACAGGTCA

11407
11415
9
reverse


 


hsa-miR-552
AACATGTGA

9513
9521
9
reverse


 


hsa-miR-552
AACAGGTTA

2441
2449
9
reverse


 


hsa-miR-552
AACAGGTAA

1569
1577
9
reverse


 


hsa-miR-583
AAAAGAGGA

2921
2929
9
reverse


 


hsa-miR-583
CAAATAGGA

2833
2841
9
reverse


 


hsa-miR-583
CAACGAGGA

1824
1832
9
reverse


 


hsa-miR-583
CAAAGAAGA

1139
1147
9
reverse


 


hsa-miR-593-3p
TGTCTCTGT

8204
8212
9
reverse


 


hsa-miR-593-3p
TGGCTCTGC

6852
6860
9
reverse


 


hsa-miR-593-3p
TGCCTCTGC

231
239
9
reverse


 


hsa-miR-593-5p
AGGCACCAG

2090
2098
9
reverse


 


hsa-miR-593-5p
AGGCACCAG

2083
2091
9
reverse


 


hsa-miR-598
ACGTCATC

11432
11439
8
reverse


 


hsa-miR-611
GCGAGGTCTC
250
4779
4788
10
reverse


 


hsa-miR-611
GAGAGGCCCC
251
2121
2130
10
reverse


 


hsa-miR-611
GAGAGGACCT
252
1546
1555
10
reverse


 


hsa-miR-616-5p
ACTCTAAAC

14510
14518
9
reverse


 


hsa-miR-619
GACCTGGA

5824
5831
8
reverse


 


hsa-miR-620
ATGAATATAG
253
14560
14569
10
reverse


 


hsa-miR-620
ATGGAAATAT
254
12111
12120
10
reverse


 


hsa-miR-620
TTGGATATAG
255
11026
11035
10
reverse


 


hsa-miR-620
GTGGAGATGG
256
10397
10406
10
reverse


 


hsa-miR-620
ATGGAGATCC
257
6268
6277
10
reverse


 


hsa-miR-620
ATGGAGGGAG
258
5626
5635
10
reverse


 


hsa-miR-620
CTGGAGAAAG
259
3827
3836
10
reverse


 


hsa-miR-620
ATCCAGATAG
260
2959
2968
10
reverse


 


hsa-miR-620
ATGGGGCTAG
261
2843
2852
10
reverse


 


hsa-miR-620
AGGGAGAGAG
262
1551
1560
10
reverse


 


hsa-miR-620
CAGGAGATAG
263
1430
1439
10
reverse


 


hsa-miR-620
TTGGAGAGAG
264
1201
1210
10
reverse


 


hsa-miR-623
TCCCTTGC

8306
8313
8
reverse


 


hsa-miR-623
TCCCTTGC

5004
5011
8
reverse


 


hsa-miR-631
CACCTGGCC

9900
9908
9
reverse


 


hsa-miR-631
GACATGGCC

8632
8640
9
reverse


 


hsa-miR-634
AACCAGCAC

4520
4528
9
reverse


 


hsa-miR-636
TGTGCTTG

10386
10393
8
reverse


 


hsa-miR-638
ACGGAGCGCG
265
4905
4914
10
reverse


 


hsa-miR-638
AGGGAGGGCG
266
4615
4624
10
reverse


 


hsa-miR-642a-5p
ATCCCTCTC

8983
8991
9
reverse


 


hsa-miR-642a-5p
GTCCCTCCC

4722
4730
9
reverse


 


hsa-miR-643
ACATGCATGC
267
15553
15562
10
reverse


 


hsa-miR-643
CCTTGTAGGC
268
15378
15387
10
reverse


 


hsa-miR-643
TCTTGTATTC
269
14423
14432
10
reverse


 


hsa-miR-643
ACTGGTATGT
270
13933
13942
10
reverse


 


hsa-miR-643
ACTTCTATTC
271
12886
12895
10
reverse


 


hsa-miR-643
ACTTTTCTGC
272
12044
12053
10
reverse


 


hsa-miR-643
GCTTGTAAGC
273
11698
11707
10
reverse


 


hsa-miR-643
AGTTGTATGT
274
10531
10540
10
reverse


 


hsa-miR-643
ACTTGGAAGC
275
8105
8114
10
reverse


 


hsa-miR-643
ACTTGTGTGG
276
7227
7236
10
reverse


 


hsa-miR-643
ACTTGTTTGA
277
1880
1889
10
reverse


 


hsa-miR-643
ACATGTTTGC
278
1695
1704
10
reverse


 


hsa-miR-650
AGGAGGCAC
279
9647
9655
9
reverse


 


hsa-miR-650
AGAAGGCAG
280
6917
6925
9
reverse


 


hsa-miR-650
AGGAGCCAG
281
3474
3482
9
reverse


 


hsa-miR-650
ATGAGGCAG
282
3052
3060
9
reverse


 


hsa-miR-651
TCATGATAAG
283
15700
15709
10
reverse


 


hsa-miR-651
TTAGGTTAAA
284
13993
14002
10
reverse


 


hsa-miR-651
TTAAAATAAG
285
13988
13997
10
reverse


 


hsa-miR-651
TTAGCATAAC
286
12788
12797
10
reverse


 


hsa-miR-651
TTATGATGAG
287
12617
12626
10
reverse


 


hsa-miR-651
TTTGGATGAG
288
11069
11078
10
reverse


 


hsa-miR-651
TGAGTATAAG
289
10767
10776
10
reverse


 


hsa-miR-651
TTACAATAAG
290
10546
10555
10
reverse


 


hsa-miR-651
TAAGGATAAA
291
8265
8274
10
reverse


 


hsa-miR-651
TGTGGATAAG
292
7222
7231
10
reverse


 


hsa-miR-651
GTAGGATAGG
293
5553
5562
10
reverse


 


hsa-miR-651
CTAGGAAAAG
294
2823
2832
10
reverse


 


hsa-miR-651
CTATGATAAG
295
1635
1644
10
reverse


 


hsa-miR-651
TAAGGATAGG
296
1562
1571
10
reverse


 


hsa-miR-654-3p
TATGTATACT
297
15493
15502
10
reverse


 


hsa-miR-654-3p
TATCTCTTCT
298
14775
14784
10
reverse


 


hsa-miR-654-3p
TCTATCTGCT
299
8354
8363
10
reverse


 


hsa-miR-654-3p
AATGTCTGGT
300
6720
6729
10
reverse


 


hsa-miR-654-3p
TATGTTTCCT
301
6638
6647
10
reverse


 


hsa-miR-654-3p
TTTTTCTGCT
302
6586
6595
10
reverse


 


hsa-miR-654-3p
TATGTCTTTT
303
6534
6543
10
reverse


 


hsa-miR-654-3p
TATATCTGCA
304
6214
6223
10
reverse


 


hsa-miR-654-3p
TATGTAGGCT
305
97
106
10
reverse


 


hsa-miR-655
GTAATACAT

15593
15601
9
reverse


 


hsa-miR-655
ATAGTACAT

4200
4208
9
reverse


 


hsa-miR-655
ATAAGACAT

3642
3650
9
reverse


 


hsa-miR-655
ATAATACAG

2265
2273
9
reverse


 


hsa-miR-655
ACAATACAT

1757
1765
9
reverse


 


hsa-miR-656
AATATTATA

657
665
9
reverse


 


hsa-miR-664-3p
TATTCATTT

9385
9393
9
reverse


 


hsa-miR-765
TGGAGGA

5020
5026
7
reverse


 


hsa-miR-766
CTCCAGCCCC
306
12901
12910
10
reverse


 


hsa-miR-766
CTCCAGCCCC
307
5032
5041
10
reverse


 


hsa-miR-767-3p
CCTGCTCAT

14871
14879
9
reverse


 


hsa-miR-767-3p
TCTTCTCAT

9155
9163
9
reverse


 


hsa-miR-875
CCTGGAAATA
308
5820
5829
10
reverse


 


hsa-miR-875
CCTAGAAACA
309
5294
5303
10
reverse


 


hsa-miR-876
TGGATTTCT

6366
6374
9
reverse


 


hsa-miR-876
TGGATTTCT

142
150
9
reverse


 


hsa-miR-888-3p
GACTGACTCC
310
15772
15781
10
reverse


 


hsa-miR-888-3p
GACTGACAGC
311
9119
9128
10
reverse


 


hsa-miR-890
TACTTGGAAG
312
8106
8115
10
reverse


 


hsa-miR-940
AAGGCAGTG

1807
1815
9
reverse


 


hsa-miR-941
CACCCAGGT

14396
14404
9
reverse


 


hsa-miR-941
CACCCTGCC

13715
13723
9
reverse


 


hsa-miR-941
CACCCCTCT

13128
13136
9
reverse


 


hsa-miR-941
CACTCAGCT

12289
12297
9
reverse


 


hsa-miR-941
CTCCCGGGT

10102
10110
9
reverse


 


hsa-miR-941
CAGCCTGCT

10034
10042
9
reverse


 


hsa-miR-941
CACCCACCT

9904
9912
9
reverse


 


hsa-miR-941
CACCTGGCC

9900
9908
9
reverse


 


hsa-miR-941
CATCTGGCT

8219
8227
9
reverse


 


hsa-miR-941
CACTCGACT

8148
8156
9
reverse


 


hsa-miR-941
CTCCCAGCT

6840
6848
9
reverse


 


hsa-miR-941
CTCACGGCT

6031
6039
9
reverse


 


hsa-miR-941
CAGCCCGCT

5928
5936
9
reverse


 


hsa-miR-941
CACCTGACT

5510
5518
9
reverse


 


hsa-miR-941
CACGCCGCT

5142
5150
9
reverse


 


hsa-miR-941
CTCCCTGCT

3983
3991
9
reverse


 


hsa-miR-941
CACCAGGCA

2087
2095
9
reverse


 


hsa-miR-941
CTCCCGGGT

390
398
9
reverse


 


hsa-miR-941
CACCCAGCC

186
194
9
reverse


 


hsa-miR-941-2
ATCCGACTGT
313
9657
9666
10
reverse


 


hsa-miR-941-2
TCCCTGCTGT
314
8726
8735
10
reverse


 


hsa-miR-941-2
TCCCAGCTGT
315
6838
6847
10
reverse


 


hsa-miR-941-2
AGCCCGCTGT
316
5926
5935
10
reverse


 


hsa-miR-941-2
ACCCGGGCGT
317
4764
4773
10
reverse


 


hsa-miR-1179
AAGTATCCTTT
318
15346
15356
11
reverse


 


hsa-miR-1179
ATGCATTCTGT
319
3357
3367
11
reverse


 


hsa-miR-1179
ATGCATTCTCT
320
1854
1864
11
reverse


 


hsa-miR-1207-5p
TGGCAGGG

11441
11448
8
reverse


 


hsa-miR-1224-3p
CTCCACCTCC
321
399
408
10
reverse


 


hsa-miR-1228-3p
TCCCACCTG

13637
13645
9
reverse


 


hsa-miR-1228-3p
TCACGCCTG

4992
5000
9
reverse


 


hsa-miR-1231
GTGTCTGGC

12807
12815
9
reverse


 


hsa-miR-1231
GTGTCCGGG

4739
4747
9
reverse


 


hsa-miR-1245
AAGTGATCT

8341
8349
9
reverse


 


hsa-miR-1245
AAGTGATCT

2020
2028
9
reverse


 


hsa-miR-1249
CGCCCTTC

5907
5914
8
reverse


 


hsa-miR-1251
ACTCTAGGT

12854
12862
9
reverse


 


hsa-miR-1251
ACTCTATCT

8357
8365
9
reverse


 


hsa-miR-1251
ACTCCAGCT

4044
4052
9
reverse


 


hsa-miR-1251
AGTCTAGCT

457
465
9
reverse


 


hsa-miR-1252
AGAGGGAAAT
322
3819
3828
10
reverse


 


hsa-miR-1252
GGAAGGAAAT
323
1625
1634
10
reverse


 


hsa-miR-1268
CGGGCGTGG

4762
4770
9
reverse


 


hsa-miR-1270
CTGGAAATA

5820
5828
9
reverse


 


hsa-miR-1270
CTGGAGATG

5055
5063
9
reverse


 


hsa-miR-1270
CTGGAGAAA

3828
3836
9
reverse


 


hsa-miR-1270
CAGGAGATA

1431
1439
9
reverse


 


hsa-miR-1272
GATGATGA

10622
10629
8
reverse


 


hsa-miR-1275
GTAGGGGAGA
324
1189
1198
10
reverse


 


hsa-miR-1302
ATGGGACACA
325
15021
15030
10
reverse


 


hsa-miR-1302
TTTGGATATA
326
11027
11036
10
reverse


 


hsa-miR-1302
TTAGGGCATA
327
8421
8430
10
reverse


 


hsa-miR-1302
TTGGAACAGA
328
6076
6085
10
reverse


 


hsa-miR-1302
CTGGGACTTA
329
4819
4828
10
reverse


 


hsa-miR-1302
GTGGGAAATA
330
3845
3854
10
reverse


 


hsa-miR-1302
TTGTGAGATA
331
1944
1953
10
reverse


 


hsa-miR-1302
CTGGGAAATA
332
867
876
10
reverse


 


hsa-miR-1324
TCAAGACAGA
333
9426
9435
10
reverse


 


hsa-miR-1827
TGAGGCAGT

3051
3059
9
reverse


 


hsa-miR-1911-3p
CACCAGGCA

2087
2095
9
reverse


 


hsa-miR-1915
CCCCAGGG

5111
5118
8
reverse


 


hsa-miR-2909
TTTAGGGCC

3728
3736
9
reverse









B. Another Exemplary Multiple miRNAs-One mRNA Paradigm Involves UCP2.
UCP2 is a mitochondrial transporter protein expressed in WAT, skeletal muscle, pancreatic islets and the central nervous system. Like UCP1, it creates proton leaks across the inner mitochondrial membrane, thus uncoupling oxidative phosphorylation from ATP synthesis (adaptive thermogenesis, see FIG. 5) (Lowell et al., Nature (2000)).
Two recent meta-analyses report an association between polymorphisms in the promoter region of UCP2 and obesity (Liu et al., Gene (2013); Andersen et al., Int J. Obes. (2013)). The first meta-analysis included 14 studies (7,647 cases and 11,322 controls) and concluded that there is a significant association of the A allele of the UCP2-866G/A polymorphism with reduced risk of obesity, especially in European populations. In the second meta-analysis including 12,984 subjects, the common UCP2-866G allele is associated with obesity. The same UCP2-866G allele is associated with decreased insulin sensitivity in 17,636 Danish subjects. In a study, UCP2 mRNA levels in visceral fat were decreased in subjects with the GG phenotype (Esterbauer et al., Nat. Genet. (2001)). A trend toward a negative correlation between subcutaneous adipocyte UCP2 mRNA and percent body fat was found in another study (Wang et al., American Journal of Physiol. (2004)). This information supports targeting UCP2 expression and activity as a meaningful way to alter adaptive thermogenesis and consequently treat human obesity. Many strategies could be implemented to achieve this goal, however, the one employed in the methods of the invention uses miRNA agents to modulate simultaneously several elements within the thermogenic pathways to increase UCP2 synthesis and activity. Both direct and indirect interactions between miRNAs and the UCP2 gene are considered. Direct interaction means the direct binding of miRNAs to the various regions of the UCP2 gene, resulting in alterations of the transcription, translation, stability and/or degradation of the UCP1 mRNA. Indirect interaction means that miRNAs alter the transcription, translation, stability and/or degradation of thermogenic mRNAs, whose expressed proteins alter the transcription of the UCP2 gene. Furthermore, indirect interaction means that miRNAs alter the transcription, translation, stability and/or degradation of other miRNAs that modify the transcription of the UCP2 gene.
The promoter region of the human UCP2 gene (ENSG00000175567, Homo sapiens uncoupling protein 2 (mitochondrial, proton carrier) (UCP2), RefSeqGene on chromosome 11) is rich is regulatory element motifs:










UCP2 Gene Regulatory Elements:















1. RXR/T3RE Motif: AGGTCA


Eight


Length: 6, Interval: 1,074 -> 1,079; 3,083 -> 3,088; 3,239 -> 3,244; 4,304 -> 4,309; 


6,965 -> 6,970; 7,420 -> 7,425; 7,677 -> 7,682; 13,319 -> 13,324; Mismatches: 0.


2. GC Box 1 Motif: CGCCC


Sixteen


Length: 5, Interval: 2,605 -> 2,609; 4,323 -> 4,327; 4,523 -> 4,527; 4,933 -> 4,937; 


4,959 -> 4,963; 5,048 -> 5,052; 5,066 -> 5,070; 5,146 -> 5,150; 5,155 -> 5,159; 


5,387 -> 5,391; 5,483 -> 5,487; 6,067 -> 6,071; 8,523 -> 8,527; 9,790 -> 9,794; 


10,819 -> 10,823; 11,754 -> 11,758; Mismatches: 0.


3. GC Box 2 Motif: GCGGG


Five


Length: 5, Interval: 4,263 -> 4,267; 4,757 -> 4,761; 4,860 -> 4,864; 7,619 -> 7,623; 


11,262 -> 11,266; Mismatches: 0.


4. GT Box 1 Motif: CACCC


Thirty


Length: 5, Interval: 1,421 -> 1,425; 1,677 -> 1,681; 1,761 -> 1,765; 1,825 -> 1,829; 


1,833 -> 1,837; 2,036 -> 2,040; 3,003 -> 3,007; 4,903 -> 4,907; 4,947 -> 4,951; 5,210 -> 


5,214; 6,204 -> 6,208; 6,247 -> 6,251; 6,469 -> 6,473; 6,828 -> 6,832; 7,681 -> 7,685; 


8,048 -> 8,052; 8,437 -> 8,441; 8,572 -> 8,576; 8,599 -> 8,603; 8,702 -> 8,706; 11,077 -> 


11,081; 11,235 -> 11,239; 12,006 -> 12,010; 12,374 -> 12,378; 13,475 -> 13,479; 13,666 -> 


13,670; 13,687 -> 13,691; 13,838 -> 13,842; 14,410 -> 14,414; 14,545 -> 14,549; 


Mismatches: 0.


5. GT Box 2 Motif: GTGGG


Twenty Six


Length: 5, Interval: 123 -> 127; 1,006 -> 1,010; 2,105 -> 2,109; 4,562 -> 4,566; 5,793 -> 


5,797; 6,029 -> 6,033; 6,034 -> 6,038; 6,040 -> 6,044; 6,150 -> 6,154; 7,271 -> 7,275; 


7,392 -> 7,396; 9,040 -> 9,044; 9,697 -> 9,701; 10,227 -> 10,231; 10,238 -> 10,242; 


10,247 -> 10,251; 11,817 -> 11,821; 12,410 -> 12,414; 12,414 -> 12,418; 12,678 -> 12,682; 


13,047 -> 13,051; 13,238 -> 13,742; 13,743 -> 13,747; 14,252 -> 14,256; 14,969 -> 14,973; 


15,104 -> 15,108; Mismatches: 0.


6. CpG Methylation Island Motif: CG


Two hundred and Ninety Five, including many between positions 4,071 to 5,212.









FIG. 8B depicts the location of these various regulatory elements in reference to the UCP2 transcription start site at nucleotide position 5,001 of the 15,174 base pair human UCP2 gene. Direct or indirect activation or repression of these regulatory elements by miRNAs will result in alterations of UCP2 gene expression and activity.
A survey of miRNAs targeting the human UCP2 3′UTR with several prediction programs, using the UCP2 Ensembl 2,113 base pair transcript ENST00000310473 as a target revealed binding sites for 161 miRNAs as shown in Table 9.







TABLE 9





miRNAs with predicted binding sites in


the 3′UTR of UCP2 transcript sequence.



















hsa-miR-1
hsa-miR-1302-5
hsa-miR-23b



hsa-miR-1-2
hsa-miR-1302-6
hsa-miR-24-1



hsa-miR-101-1
hsa-miR-1302-7
hsa-miR-24-2



hsa-miR-101-2
hsa-miR-1302-8
hsa-miR-27b-5p



hsa-miR-103
hsa-miR-1302-9
hsa-miR-28



hsa-miR-105-1
hsa-miR-1303
hsa-miR-296-3p



hsa-miR-105-2
hsa-miR-130a
hsa-miR-296-5p



hsa-miR-106b
hsa-miR-1321
hsa-miR-3064



hsa-miR-107
hsa-miR-138-1
hsa-miR-323a



hsa-miR-1204
hsa-miR-138-2
hsa-miR-328



hsa-miR-1207
hsa-miR-149
hsa-miR-330



hsa-miR-1208
hsa-miR-150-3p
hsa-miR-331



hsa-miR-1226
hsa-miR-150-5p
hsa-miR-338



hsa-miR-1246
hsa-miR-1538
hsa-miR-342



hsa-miR-1252
hsa-miR-155
hsa-miR-3619



hsa-miR-1253
hsa-miR-15a
hsa-miR-370



hsa-miR-1255a
hsa-miR-15b
hsa-miR-377



hsa-miR-1255b-1
hsa-miR-16-1
hsa-miR-378a



hsa-miR-1255b-2
hsa-miR-16-2
hsa-miR-383



hsa-miR-1260a
hsa-miR-184
hsa-miR-411



hsa-miR-1262
hsa-miR-185-3p
hsa-miR-412



hsa-miR-1263
hsa-miR-185-5p
hsa-miR-422a



hsa-miR-1265
hsa-miR-186
hsa-miR-424



hsa-miR-1275
hsa-miR-188
hsa-miR-425



hsa-miR-1276
hsa-miR-18a
hsa-miR-4291



hsa-miR-1278
hsa-miR-18b
hsa-miR-432-3p



hsa-miR-1285-1
hsa-miR-193a
hsa-miR-4505



hsa-miR-1286
hsa-miR-195
hsa-miR-450b



hsa-miR-1293
hsa-miR-199b
hsa-miR-453



hsa-miR-1300
hsa-miR-200a
hsa-miR-4533



hsa-miR-1302-1
hsa-miR-203
hsa-miR-4539



hsa-miR-1302-10
hsa-miR-206
hsa-miR-4745



hsa-miR-1302-11
hsa-miR-214
hsa-miR-4747



hsa-miR-1302-2
hsa-miR-219-1
hsa-miR-485-5p



hsa-miR-1302-3
hsa-miR-219-2
hsa-miR-486



hsa-miR-1302-4
hsa-miR-221-5p
hsa-miR-490



hsa-miR-491
hsa-miR-584
hsa-miR-663b



hsa-miR-493
hsa-miR-608
hsa-miR-664-5p



hsa-miR-497
hsa-miR-612
hsa-miR-675



hsa-miR-498
hsa-miR-613
hsa-miR-7-1



hsa-miR-503
hsa-miR-615-3p
hsa-miR-7-2



hsa-miR-505
hsa-miR-618
hsa-miR-7-3



hsa-miR-508-3p
hsa-miR-625
hsa-miR-708



hsa-miR-532
hsa-miR-626
hsa-miR-761



hsa-miR-539
hsa-miR-634
hsa-miR-765



hsa-miR-541
hsa-miR-635
hsa-miR-769



hsa-miR-5481
hsa-miR-638
hsa-miR-770



hsa-miR-552
hsa-miR-645
hsa-miR-876



hsa-miR-563
hsa-miR-646
hsa-miR-877



hsa-miR-575
hsa-miR-647
hsa-miR-921



hsa-miR-577
hsa-miR-652
hsa-miR-922



hsa-miR-580
hsa-miR-654
hsa-miR-92a-1



hsa-miR-583
hsa-miR-658
hsa-miR-92a-2-5p




hsa-miR-663a
hsa-miR-92b










Moreover, a survey of miRNAs targeting the human UCP2 5′UTR with several prediction programs, using the human UCP2 gene (ENSG00000175567, 15,174 base pair (bp) of the, including 5,000 bp 5′UTR as a target revealed binding sites for 54 miRNAs in UCP2 5′UTR as shown in Table 10.







TABLE 10







miRNAs with predicted binding sites in the 5′UTR of UCP2 gene sequence.














Seed


SEQ ID




MicroRNA
Length
Start
Sequence
NO
End
P value
















hsa-let-7c
9
3052
UAGAGUUAC

3044
0.0374


 


hsa-let-7i-3p
9
3051
CUGCGCAAG

3043
0.0374


 


hsa-miR-1228-5p
9
3419
UGGGCGGGG

3411
0.0374


 


hsa-miR-1229-3p
9
3419
UCUCACCAC

3411
0.0374


 


hsa-miR-129-1-3p
10
2784
AGCCCUUACC
334
2775
0.0095


 


hsa-miR-1302
9
4219
UGGGACAUA

4211
0.0374


 


hsa-miR-1303
9
2159
UUAGAGACG

2151
0.0374


 


hsa-miR-136
9
4486
CUCCAUUUG

4478
0.0374


 


hsa-miR-155
9
2160
UUAAUGCUA

2152
0.0374


 


hsa-miR-16
10
3603
UAGCAGCACG
335
3594
0.0095


 


hsa-miR-18a-3p
10
3603
ACUGCCCUAA
336
3594
0.0095


 


hsa-miR-190
9
2428
UGAUAUGUU

2420
0.0374


 


hsa-miR-191
9
3052
CAACGGAAU

3044
0.0374


 


hsa-miR-192
9
4390
CUGACCUAU

4382
0.0374


 


hsa-miR-194
9
1643
UGUAACAGC

1635
0.0374


 


hsa-miR-197
9
5001
UCACCACCU

4993
0.0374


 


hsa-miR-19b-2-5p
10
3052
AGUUUUGCAG
337
3043
0.0095


 


hsa-miR-203
9
3051
UGAAAUGUU

3043
0.0374


 


hsa-miR-218
10
3603
UUGUGCUUGA
338
3594
0.0095


 


hsa-miR-218-1-3p
9
5001
UGGUUCCGU

4993
0.0374


 


hsa-miR-219-1-3p
9
3614
AGAGUUGAG

3606
0.0374


 


hsa-miR-26a-2-3p
9
2163
CCUAUUCUU

2155
0.0374


 


hsa-miR-27a-3p
10
3603
UUCACAGUGG
339
3594
0.0095


 


hsa-miR-27a-5p
11
3336
AGGGCUUAGCU
340
3326
0.0024


 


hsa-miR-28-5p
10
3603
AAGGAGCUCA
341
3594
0.0095


 


hsa-miR-331-3p
9
4134
GCCCCUGGG

4126
0.0374


 


hsa-miR-337-5p
10
115
GAACGGCUUC
342
106
0.0095


 


hsa-miR-340-3p
9
1872
CCGUCUCAG

1864
0.0374


 


hsa-miR-34c-3p
11
2162
AAUCACUAACC
343
2152
0.0024


 


hsa-miR-373-5p
11
530
ACUCAAAAUGG
344
520
0.0024


 


hsa-miR-425
9
1013
AAUGACACG

1005
0.0374


 


hsa-miR-497
9
3661
AGCAGCACA

3653
0.0374


 


hsa-miR-501-5p
9
4164
AUCCUUUGU

4156
0.0374


 


hsa-miR-505
9
1015
GUCAACACU

1007
0.0374


 


hsa-miR-508-3p
9
1274
GAUUGUAGC

1266
0.0374


 


hsa-miR-509-3p
12
2554
UGAUUGGUACGU
345
2543
0.0006


 


hsa-miR-512-5p
10
987
ACUCAGCCUU
346
978
0.0095


 


hsa-miR-514
9
5001
UUGACACUU

4993
0.0374


 


hsa-miR-515-5p
9
59
UUCUCCAAA

51
0.0374


 


hsa-miR-518a-3p
9
19
GAAAGCGCU

11
0.0374


 


hsa-miR-519e-5p
11
2525
UCUCCAAAAGG
347
2515
0.0024


 


hsa-miR-548a-3p
10
680
CAAAACUGGC
348
671
0.0095


 


hsa-miR-550a-3p
9
4312
GUCUUACUC

4304
0.0374


 


hsa-miR-571
9
739
UGAGUUGGC

731
0.0374


 


hsa-miR-578
9
1377
CUUCUUGUG

1369
0.0374


 


hsa-miR-606
9
4420
AACUACUGA

4412
0.0374


 


hsa-miR-615-5p
10
1140
GGGGGUCCCC
349
1131
0.0095


 


hsa-miR-638
9
2710
GGGAUCGCG

2702
0.0374


 


hsa-miR-657
12
1316
GCAGGUUCUCAC
350
1305
0.0006


 


hsa-miR-658
9
3673
GGCGGAGGG

3665
0.0374


 


hsa-miR-877-3p
9
4349
UCCUCUUCU

4341
0.0374


 


hsa-miR-93-3p
9
799
ACUGCUGAG

791
0.0374


 


hsa-miR-96-3p
9
799
AAUCAUGUG

791
0.0374


 


hsa-miR-99b-3p
9
2163
CAAGCUCGU

2155
0.0374









c) A Multiple microRNAs-Multiple mRNAs Paradigm.

The 83 thermogenic regulator molecules selected in Table 1 were screened for high stringency Multiple miRNAs-Multiple mRNAs associations. The results of these analyses with 7 major prediction tools are shown in FIG. 4. The union of these 7 tools produces 4439 miRNA-gene couples. Overlap between these tools decreases as the number of tools increases, reaching only 15 miRNA-gene couples when 7 tools are considered.
d) An Over-Representation of One microRNA Seed Sequence Motif Among Co-Regulated mRNA Targets Paradigm.
Several approaches can be used to identify pathway-specific miRNAs. For example, searching the 3′-UTRs of putatively co-regulated genes for an over-represented sequence from a miRNA seed region could identify a common regulatory miRNA. To determine if particular miRNA seed sequences were overrepresented among the 3′ UTR of the chosen 83 thermogenesis targets, the miRvestigator web application (mirvestigator. systemsbiology.net/) was employed. Using the following parameters (motif size of 8 bp, default Weeder model, seed model of 8mer, 100% complementarity homology and 0.25 wobble base-pairing allowed), it was determined that that the motif 5′-UUUGUACA-3′ recognized by hsa-miR-19a/-19b is overrepresented among 15 of the 83 thermogenesis targets with a complementarity p value of 1.7×10−04 as shown in Table 11. Of note is that hsa-miR-19 has been reported as an abundant adipocyte miRNA.







TABLE 11







Complementarity between the common motif UUUGUACA and hsa-miR-


19a/19b.















Length of

Complementarity


miRNA Name
miRNA Seed
Seed Model
Complementarity
Complementary Base-Pairing
P-Value





hsa-miR-19a hsa-miR-19b
UGUGCAAA
8mer
8




1.7e-04









The Minimum Free Energy levels of the hsa-miR-19 mRNA/miRNA duplexes identified by miRvestigator were quite low, favoring tight binding. Accordingly, the miRvestigator analysis was repeated with less stringent levels of complementarity. This analysis identified a further 10 additional targets (CEBPD, PRKAA1, TWIST1, IRS1, NCOA1, NCOA2, NCOA3, KLF5, RPS6KB1, NRIP1) with 95% similarity to the consensus hsa-miR-19 motif. Interestingly, hsa-miR-19 is among the most abundant miRNAs in adipose tissue. The genes identified as containing a sequence complementary to hsa-miR-19 seed region are set forth in Table 12.







TABLE 12







Thermogenic regulators identified as


targets for hsa-miR-19.















 
%







Similarity
Minimum






to
Free





Start
Consensus
Energy





Relative
Motif
(MFE) of




Sequence
to Stop
(Quality =
mRNA-



Gene
of
Codon
High|
miRNA


Gene
symbol
Site
(bp)
Medium|Fair
Duplex















650
BMP2
UUUGUACA
386
100.00
−6.80


 


1052
CEBPD
UUUGUAAA
263
95.44
−3.40


 


7132
TNFRSF1A
UUUGUACA
510
100.00
−6.80


 


5562
PRKAA1
UUUGUAAA
2400
95.44
−3.40


 


5563
PRKAA2
UUUGUACA
542
100.00
−6.80


 


655
BMP7
UUUGUACA
1927
100.00
−6.80


 


652
BMP4
UUUGUACA
770
100.00
−6.80


 


133522
PPARGC1B
UUUGUACA
7199
100.00
−6.80


 


7474
WNT5A
UUUGUACA
1414
100.00
−6.80


 


6720
SREBF1
UUUGUACA
510
100.00
−6.80


 


7291
TWIST1
UUUGUAAA
649
95.44
−3.40


 


3667
IRS1
UUUGUAAA
992
95.44
−3.40


 


10499
NCOA2
UUUGUAAA
1381
95.44
−3.40


 


8204
NRIP1
UUUGUACA
1718
100.00
−6.80


 


8204
NRIP1
UUUGUAAA
1935
95.44
−3.40


 


8202
NCOA3
UUUGUAAA
965
95.44
−3.40


 


1385
CREB1
UUUGUAAA
1973
95.44
−3.40


 


1385
CREB1
UUUGUACA
2822
100.00
−6.80


 


1385
CREB1
UUUGUACA
2822
100.00
−6.80


 


1385
CREB1
UUUGUAAA
4175
95.44
−3.40


 


3643
INSR
UUUGUAAA
2105
95.44
−3.40


 


8013
NR4A3
UUUGUACA
2347
100.00
−6.80


 


860
RUNX2
UUUGUACA
2425
100.00
−6.80


 


6776
STAT5A
UUUGUACA
1214
100.00
−6.80


 


1874
E2F4
UUUGUACA
755
100.00
−6.80


 


688
KLF5
UUUGUAAA
549
95.44
−3.40


 


8648
NCOA1
UUUGUAAA
381
95.44
−3.40


 


6198
RPS6KB1
UUUGUAAA
2531
95.44
−3.40









Accordingly, the miRvestigator analysis was repeated with less stringent levels of complementarity (motif size of 8 bp, default Weeder model, seed model of 8mer, 95% complementarity homology and 0.25 wobble base-pairing allowed). This analysis identified a further 10-12 additional targets (CEBPD, CREB1, PRKAA1, TWIST1, INSR, IRS1, NCOA1, NCOA2, NCOA3, KLF5, RPS6 KB1, NRIP1) with 95% similarity to the consensus hsa-miR-19 motif. Interestingly, hsa-miR-19 is among the most abundant miRNAs in adipose tissue. The genes identified as containing a sequence complementary to hsa-miR-19 seed region are set forth in Table 13.







TABLE 13 







Thermogenic regulators identified as


targets for hsa-miR-19a/b with 95% to 100%


similarity to consensus motif.















 
%
Minimum






Similarity
Free





Start
to
Energy





Relative
Consensus
(MFE)





to
Motif
of




Sequence
Stop
(Quality =
mRNA-



Gene
of
Codon
High|
miRNA


Gene
symbol
Site
(bp)
Medium|Fair
Duplex















650
BMP2
UUUGUACA
386
100.00
−6.80


 


1052
CEBPD
UUUGUAAA
263
95.44
−3.40


 


7132
TNFRSF1A
UUUGUACA
510
100.00
−6.80


 


4040
LRP6
UUUGUACA
151
100.00
−6.80


 


4040
LRP6
UUUGUAAA
4965
95.42
−3.40


 


5562
PRKAA1
UUUGUAAA
2400
95.42
−3.40


 


5563
PRKAA2
UUUGUACA
542
100.00
−6.80


 


655
BMP7
UUUGUACA
1927
100.00
−6.80


 


652
BMP4
UUUGUACA
770
100.00
−6.80


 


133522
PPARGC1B
UUUGUACA
7199
100.00
−6.80


 


1874
E2F4
UUUGUACA
755
100.00
−6.80


 


7474
WNT5A
UUUGUACA
1414
100.00
−6.80


 


6720
SREBF1
UUUGUACA
510
100.00
−6.80


 


7291
TWIST1
UUUGUAAA
649
95.44
−3.40


 


3667
IRS1
UUUGUAAA
992
95.42
−3.40


 


10499
NCOA2
UUUGUAAA
1381
95.42
−3.40


 


8204
NRIP1
UUUGUACA
1718
100.00
−6.80


 


8204
NRIP1
UUUGUAAA
1935
95.42
−3.40


 


8202
NCOA3
UUUGUAAA
965
95.42
−3.40


 


3643
INSR
UUUGUAAA
2105
95.42
−3.40


 


8013
NR4A3
UUUGUACA
2347
100.00
−6.80


 


6776
STAT5A
UUUGUACA
1214
100.00
−6.80


 


688
KLF5
UUUGUAAA
549
95.42
−3.40


 


8648
NCOA1
UUUGUAAA
381
95.42
−3.40


 


860
RUNX2
UUUGUACA
2425
100.00
−6.80


 


1385
CREB1
UUUGUAAA
1973
95.42
−3.40


 


1385
CREB1
UUUGUACA
2822
100.00
−6.80


 


1385
CREB1
UUUGUAAA
4175
95.42
−3.40


 


6198
RPS6KB1
UUUGUAAA
2531
95.42
−3.40









Without wobbling, the same motif 5′-UUUGUACA-3′ is overrepresented among targets of hsa-miR-1283 with a complementarity p value of 1.4×10−4. Furthermore, hsa-miR-1283 binds to other mRNAs of interest like ABCA1 (cholesterol transporter), the adiponectin receptor and the transcription factor TCF7L2 that is implicated in genetic human obesity.

Similarly, other miRNA over-represented seed sequences were identified for miRNAs expressed in adipocytes. They include the universal hsa-let-7 family (sequence CUAUACAA, p value=7.5e-04) and the adipocyte-rich hsa-miR-30 family (sequence UGUAAACA, p value=1.9×10−3) to name a few.
With respect to PRDM16, CIDEA, NRIP1, KDM3A, CEPPB, PPARG, PPARGC1A, and PPKAA2, which according to the STRING software package are directly linked to UCP1, it appears that all of them share (at motif size 8 bp, default weeder model, seed model 8mer, 95% complementarity homology and 0.25 wobble base-pairing allowed) a consensus sequence with several miRNAs, including hsa-miR-3658 (p value=1.9e-003) and the hsa-miR-30 family (p value=6.3e-003) as follows:




e) An Intronic miRNA-Multiple mRNAs Pathway-Specific Paradigm.

Many mammalian miRNAs are located within introns of protein-coding genes rather than in their own unique transcription units. Intronic miRNAs are typically expressed and processed with the precursor mRNA in which they reside. Although the intronic miRNAs and their host genes can be regulated independently, an intronic miRNA can down-regulate its own host protein-coding gene by targeting the host gene's UTR. Feedback regulation on host protein-coding genes could be achieved by selecting the transcription factors that are miRNA targets or by protein-protein interactions between intronic miRNA host gene product and miRNA target gene products. As an example, miR-33 acts in concert with the SREBP host genes to control cholesterol homeostasis and the pharmacological inhibition of miR-33a and miR-33b is a promising therapeutic strategy to raise plasma HDL and lower VLDL triglyceride levels for the treatment of dyslipidaemias.
Examination of the 83 thermogenic target genes reveals two intronic miRNAs: miR-378 located in the PPARGC1B gene and miR-4251 located in the PRDM16 gene.
Mining of the Internet tools predicting miRNA targets indicates that miR-378 targets include BMP2, PPARA, PPARGC1A, PRDM16, STATS and WNT10A as well as ADIPOQ and IGFR1; and that miR-4251 targets include BMP2, CTBP1, CTBP2, MAPK14, NCOA3, PLAC8, PPARA, PPARD, TRPM8, as well as ABCA5, ABCA13, ADIPOQR2, KDM5B, KLF-12, KLF-14 and TCF7L2.
Example 3
High-Content Cellular Phenotypic Screening
High-content screening methods are used to screen for novel miRNA agents that modulate the activity of thermogenic regulators (e.g., UCP1 and UCP2). High-content screening is a drug discovery method that uses images of living cells to facilitate molecule discovery. Such automated image based screening methods are particularly suitable for identifying agents which alter cellular phenotypes.
WAT cells which contain a single large lipid droplet, whereas, in contrast, BAT cells contain numerous smaller droplets and a much higher number of mitochondria, which contain iron and make them appear brown. The large number of mitochondria in BAT leads to an increased oxygen consumption, when compared to WAT. Accordingly, it is possible to distinguish between BAT and WAT cells visually based on their cellular phenotype.
Accordingly, high-content screening methods were used to screen for novel miRNA agents that modulate the activity of thermogenic regulators. Specifically, the phenotypic appearance of cultured human adipocytes and adipose tissue derived mesenchymal stem cells grown in the presence and absence of miRNA agonists or antagonists was assessed over two weeks by phase contrast microscopy of the cultured cells, measurement of the cellular lipid content (using Oil Red O Staining or Nile Red fluorescence); mitochondrial content (e.g., using Life Technologies Mito-Tracker Red FM), and/or oxygen consumption in vitro (e.g., using the Seahorse Bioscience Extra-Cellular Flux Instrument). mRNA expression is measured by targeted q-RT-PCR and universal RNA-Sequencing. Protein expression is measured by targeted Western Blotting and universal proteomic profiling.
A. Differentiation of Human Pre-Adipocytes into Adipocytes.
1. Differentiation Protocol.
In order to assess the effect of miRNA analogs on human pre-adipocytes differentiation into mature adipocytes, human subcutaneous pre-adipocytes (SuperLot 0048 from 8 female donors, ZenBio, N.C.) were plated on Day 0 into 96-well plates and allowed to attach overnight in preadipocyte medium (DMEM/Ham's F-12 (1:1, v/v), HEPES buffer, Fetal bovine serum and Antibiotics). The next day (Day 1), the medium was removed and replaced with differentiation medium (DMEM/Ham's F-12 (1:1, v/v), 100 μM Ascorbic Acid, 0.85 μM insulin, 20 nM sodium selenite, 0.2 nM, triiodothryonine, 1 μM dexamethasone, 100 μM isobutyl-methylxanthine, 100 nM Rosiglitazone and Antibiotics. The cells were allowed to incubate for 2 days at 37°, 5% CO2. After 2 days (Day 3), the medium was removed and replaced with fresh maintenance medium (DMEM/Ham's F-12 (1:1, v/v), 100 μM Ascorbic Acid, 0.85 μM insulin, 20 nM sodium selenite, 0.2 nM triiodothryonine, and Antibiotics). On Day 3, the cells were transfected with miRNA analogs (Dharmacon specific miRIDIAN Mimics and Hairpin Inhibitors) using the transfecting agent Dharmafect1. All treatments were in triplicate. Post transfection, the negative control was maintenance medium only and the positive control was maintenance medium with 100 nM of the PPARG agonist rosiglitazone. After 2 days, medium was removed and replaced with fresh maintenance medium. The maintenance medium then changed every two days until the end of the treatment period (Day 15). At the end of the treatment (total of 15 days in culture) cells were processed for Phenotyping and Genotyping Screening.
1. Transfection of Pre-Adipocytes.
Transfection reagents are used to facilitate the penetration of miRNA analogs into target cells. As an example, the extent of transfection efficiency we achieved in pre-adipocytes with the transfecting agent Dharmafect 1 (Dharmacon, Colo.) is depicted herein. Transfection efficiency was assessed in two ways:
a. Measurement of Cellular Epifluorescence after Transfection with Fluorescent miRNA Analogs.
Fluorescence was measured on Day 15 (540 excitation/590 emission) in cells transfected on Day 3 with the Dy547-labeled non-targeting miRIDIAN Mimic and Hairpin Inhibitor (100 nM). As shown in FIG. 9, there was a significantly greater fluorescence of cells transfected with the fluorescent miRNA analogs, even 12 days after transfection:
b. Reduction of Control Gene Expression.
To confirm successful transfection of pre-adipocytes, the reduction of expression of the control gene GAPDH (“housekeeping gene”) was measured 4 days (Day 7) (FIG. 10A) and 12 days (Day 15) (FIG. 10B) after transfection of pre-adipocytes with a GAPDH-specific siRNA. Cell lysates were obtained and RT-PCR was conducted using pure RNA obtained by Cells-to-Ct reagents. 91% and 86% knockdowns of the GAPDH mRNA expression were observed at Day 4 and Day 12 post transfection, both highly significant, as shown in FIG. 10.
c. Phenotypic Changes During Human Preadipocytes Differentiation into Adipocytes.
At the end of treatment (15 days in culture) cells were stained with Oil Red O for assessment of lipid content. As shown in FIG. 11, in the presence of medium without rosiglitazone, the preadipocytes show little differentiation into lipid-loaded mature adipocytes. In the presence of differentiation medium including 100 nM Rosiglitazone for 2 days followed by maintenance medium for 12 days (negative control), some differentiation into lipid-loaded mature adipocytes is noted. In the presence of 100 nM rosiglitazone throughout the experiment (positive control), most of the cells became lipid-loaded mature adipocytes. As an example, in the presence of 25 nM hsa-miR-30b mimic, about half of the cells became lipid-loaded mature adipocytes. The non targeting miRNA mimic and inhibitor showed patterns similar to the negative control.
d. Genotypic Changes During Human Pre-Adipocyte Differentiation into Adipocytes.
Profiling of mRNA changes occurring during the differentiation of human pre-adipocyte into mature adipocyte induced by rosiglitazone or miRNA analogs was performed by RNA-Seq technology. Small RNA sequencing (RNA-Seq) is a high-throughput next-generation sequencing platform which now allows transcriptome-wide profiling of all small RNAs, known and unknown, with no need for prior sequence or secondary structure information.
RNA samples were extracted from pre-adipocytes (pre-adipocyte negative control) and from pre-adipocytes cultured in the presence of 100 nM rosiglitazone (differentiation positive control) or 25 nM miRNA mimics or inhibitors for 12 days. RNA sequencing was performed on the Illumina Hi-Seq 2000 equipment. The results were mapped against Human Genome 19 (http://genome.ucsc.edu/). It appears that in the presence of a miRNA analog, between 313 and 449 mRNA are significantly differentially expressed in reference to pre-adipocytes. In reference to Rosiglitazone, the number of significantly differentially expressed genes is reduced between 111 and 216, thus suggesting common pathways of activation of adipocyte differentiation between miRNAs and the PPARG analog.
Regarding our 83 thermogenic activators and inhibitors, the expression of 73 of them is altered in the presence of rosiglitazone or miRNA analogs. The changes of mRNA expression of the thermogenesis targets in the presence of rosiglitazone (FIG. 12A) or miRNA analogs hsa-let-7a inhibitor, hsa-miR-1 mimic, hsa-miR-19b mimic, hsa-miR-30b mimic or control adipocytes are shown on FIGS. 12B-F, respectively).
Changes in mRNA expression of UCP1, 2 and 3 were also measured in the presence of rosiglitazone or miRNA analogs, as shown below in Table 14.







TABLE 14







Changes in thermogenic mRNA expression.









mRNA Expression changes (log ratios)










Agent
UCP1
UCP2
UCP3













Rosiglitazone
15.70
263
0.26


hsa-let-7a inhibitor
2.23
173
0.65


hsa-miR-1 mimic
0.41
110
0.40


hsa-miR-19b mimic
0.18
33
0.26


hsa-miR-30b mimic
0.76
119
0.28


Baseline level in pre-adipocytes
0.02
1.35
0.30









The expression levels of the three Uncoupling Proteins were low in pre-adipocytes. The expression of UCP1 was significantly increased in the presence of rosiglitazone 100 nM which was renewed with the culture medium every other day. The magnitude of UCP1 mRNA rise with the miRNA analogs was lower than with rosiglitazone, but one has to keep in mind the miRNA analogs concentration used (25 nM) and the fact that only one transfection was performed 12 days before RNA extraction. A major finding is the dramatic increase of UCP2 expression in the presence of rosiglitazone as well as the miRNA analogs. The expression of UCP3 did not change in any condition, as expected for a gene that is mainly expressed in myocytes. This increase in UCP1 and UCP2 expression suggests that administration of these miRNA produces adipocytes with greater potential for thermogenesis and thus are likely effective pharmaceuticals for the treatment of obesity and other metabolic diseases and disorders.
Furthermore, we looked at genes differentially expressed during pre-adipocyte culture in the presence of miRNA analogs. As an example shown on FIG. 16, an M-A plot was created to visualize the differences of mRNA expression between pre-adipocytes grown in maintenance medium and pre-adipocytes grown in the presence of hsa-miR-19b mimic. The x-axis is the mean gene expression and the y-axis is the difference between pairs in logarithmic scale. The red dots are the differentially expressed genes (up regulated above zero and down regulated below zero). The gray dots are the genes not differentially expressed between control and hsa-miR-19b mimic (up regulated above zero and down regulated below zero).
As an example shown on FIG. 17, in reference to pre-adipocytes cultured in maintenance medium only, the numbers of significantly differentially expressed genes in the presence of the miRNA analogs hsa-let-7a inhibitor, hsa-miR-1 mimic, hsa-miR-19b mimic and hsa-miR-30b mimic were respectively 406, 382, 370 and 433. A set of 127 genes was commonly upregulated by these 4 miRNA analogs (Venn Diagram, FIG. 17).
They include not only some of our 83 thermogenic targets like ALDH1A1, AZGP1, CEBPA, PPARGC1A, UCP1 and UCP2 highlighted in green, but also numerous genes involved in lipid metabolism and adipocyte differentiation, highlighted in yellow (Table 15).







TABLE 15





Set of 127 genes commonly 


upregulated by 4 miRNA analogs.


























A set of 60 genes was commonly downregulated by these 4 miRNA analogs (Venn Diagram, FIG. 18).
They include numerous chemokines genes and genes involved in cell proliferation and (Table 16).







TABLE 16





Set of 60 genes commonly downregulated by 4 miRNA analogs.




















ACTC1
CENPF
ID1
KRTAP2-1



ANLN
CKAP2L
ID3
MALL



ARSI
CXCL1
IER3
MMP3



ATOH8
CXCL2
IL13RA2
NCAPH



AURKB
CXCL3
IL6
PHLDA1



BLM
CXCL5
IL8
PLK1



BRCA2
CXCL6
INHBA
PPAPDC1A



BUB1
E2F7
IQGAP3
PTGS2



BUB1B
ESCO2
KIAA1244
RELN



CASC5
FAM83D
KIF11
SHCBP1



CCL26
GABBR2
KIF14
SLC17A9



CDC6
GREM2
KIF18B
SLC6A17



CDCA5
GTSE1
KIF2C
THBD



CDCA8
HAS1
KIFC1
TMSL3



CDH15
HJURP
KRT34
TOP2A










B. Differentiation of Human White Adipocytes into Brown Adipocytes.

1. Differentiation Protocol.

In order to assess the effect of miRNA analogs on human white adipocytes differentiation into brown adipocytes, human subcutaneous pre-adipocytes (SuperLot 0048 from 8 female donors, ZenBio, N.C.) were plated on Day 0 into 96-well plates and allowed to attach overnight in preadipocyte medium (DMEM/Ham's F-12 (1:1, v/v), HEPES buffer, Fetal bovine serum and Antibiotics). The next day (Day 1), the medium was removed and replaced with differentiation medium-2 (DMEM/Ham's F-12 (1:1, v/v), HEPES buffer, Fetal bovine serum, Biotin, Pantothenate, Human insulin, Dexamethasone, Isobutyl-methylxanthine, Proprietary PPARG agonist and Antibiotics. The cells were allowed to incubate for 7 days at 37° C., 5% CO2. After 7 days (Day 7), a partial medium exchange was performed with AM-1 adipocyte maintenance medium (DMEM/Ham's F-12 (1:1, v/v), HEPES buffer, Fetal bovine serum, Biotin, Pantothenate, Human insulin, Dexamethasone and Antibiotics). The cells were allowed to incubate for an additional 7 days at 37° C., 5% CO2. On Day 17, the cells were transfected with miRNA analogs (Dharmacon specific miRIDIAN Mimics and Hairpin Inhibitors) using the transfecting agent Dharmafect 3. All treatments were in triplicate. Post transfection, the negative control was maintenance medium only and the positive control was maintenance medium with 100 nM of the PPARG agonist rosiglitazone. After 2 days, medium was removed and replaced with fresh maintenance medium. The maintenance medium then changed every two to three days until the end of the treatment period (Day 30). At the end of the treatment (total of 30 days in culture) cells were processed for Phenotyping and Genotyping Screening.
2. Transfection of Adipocytes.
Transfection reagents are used to facilitate the penetration of miRNA analogs into target cells.
As an example, the extent of transfection efficiency we achieved in adipocytes with the transfecting agent Dharmafect 3 (Dharmacon, Colo.) is depicted herein. Transfection efficiency was assessed in two ways:
a. Measurement of Cellular Epifluorescence after Transfection with Fluorescent miRNA Analogs.
Fluorescence was measured on Day 30 (540 excitation/590 emission) in cells transfected on Day 17 with the Dy547-labeled non-targeting miRIDIAN Mimic and Hairpin Inhibitor (100 nM). As shown in FIG. 13, there was a significantly greater fluorescence of cells transfected with the fluorescent miRNA analogs, even 12 days after transfection:
b. Reduction of Control Gene Expression.
To confirm successful transfection of adipocytes, the reduction of expression of the control gene GAPDH (“housekeeping gene”) was measured 4 days (Day 22) and 12 days (Day 30) after Dharmafect 3 (Dharmacon, Colo.) mediated transfection of adipocytes with a GAPDH-specific siRNA. Cell lysates were obtained and RT-PCR was conducted using pure RNA obtained by Cells-to-Ct reagents. Efficient transfection of mature adipocytes (a cell type known to be difficult to transfect) was achieved with the transfecting agent Dharmafect 3.54% and 73% knockdowns of the GAPDH mRNA expression were observed at Day 4 and Day 12 post transfection, both highly significant, as shown in FIG. 14.
c. Optimization of Human Mature Adipocyte Transfection.
As efficient transfection of mature adipocytes is known to be difficult to achieve, we tested eleven different transfecting agents and assessed the degree of reduction of mRNA expression of the control gene GAPDH. Human subcutaneous pre-adipocytes were plated in 6-wll plates and differentiated for two weeks following the protocol described above. Subsequently, a miRNA mimic (50 nM) targeting GAPDH was introduced into the differentiated adipocytes using transfecting agents following their manufactures' protocol. The transfected cells were incubated for 72 hours with reagents and miRNA mimic, then switched to maintenance medium. Fourteen days post-transfection, RNA was isolated using RNeasy Mini kit and RT-PCR reactions for the control gene GAPDH and the reference gene 18S were performed in triplicate using 100 ng of cDNA per well.
The amounts of RNA extracted per well were very similar, except for the transfecting agents TransIT TKO and TransIT siQuest which may produce potential cellular toxicity in the conditions of the experiment (FIG. 19).
The cells transfected with Dharmacon 1 and siPORT NeoFX had significantly reduced levels of 18S expression and were excluded from the RT-PCR experiment analysis. Among the remaining 7 transfecting agents analyzed, the often-used transfecting agent Lipofectamine RNAiMAX led to a 66% reduction of GAPDH expression at day 14 post-transfection, Dharmafect 3 and Dharmafect 4 respectively produced 60% and 75% reduction of GAPDH expression (FIG. 20).
3. Phenotypic Changes During Maintenance of Human Adipocytes in Culture for Two More Weeks.
At the end of treatment (total of 30 days in culture) cells were stained with Oil Red O for assessment of lipid content. In the presence of medium without rosiglitazone from Day 16 to Day 30, the adipocytes appear loaded with large lipid droplets. In the presence of differentiation medium including 100 nM Rosiglitazone for 2 days followed by maintenance medium for 12 days (negative control), little change in appearance of the lipid-loaded mature adipocytes is noted. In the presence of 100 nM rosiglitazone throughout the experiment (positive control), the intensity of the red staining seems reduced. As an example, in the presence of 25 nM hsa-miR-30b mimic, the intensity of the red staining seems also reduced and the lipid droplets appear smaller.
The amount of lipids present in the mature adipocytes at Day 30 was measured with the fluorescent Nile Red Dye. As shown in FIG. 15, the highest fluorescence was noted in the adipocytes which were not exposed to rosiglitazone from Day 15 to day 30. A similar fluorescence level was noted in the cells which were transfected with the non targeting miRNA mimic and inhibitor. When the cells were exposed to rosiglitazone for two days, the fluorescence dropped significantly and was further reduced in the presence of rosiglitazone from Day 15 to Day 30. It appears that in the presence of the miRNA inhibitors tested, the level of fluorescence is within the range observed with rosiglitazone 2 day to throughout. In the presence of miRNA mimics, the level of fluorescence appears lower, an indication of lower lipid content.
Example 4
High-Throughput miRNA Target Screening by Luciferase Activity and qRT-PCR
High-throughput screening using luciferase reporter assay constructs are used to identify novel miRNA targets involved in thermogenesis.
Luciferase is commonly used as a reporter to assess the transcriptional activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of a promoter of interest. SwitchGear Genomics has created a genome-wide library of over 18,000 human promoters and 12,000 human 3′ UTR regions cloned into an optimized luciferase reporter vector system containing SwitchGear's RenSP reporter cassette (GoCloneTM) as a component of the LightSwitchTM Luciferase Assay System. This modified form of luciferase greatly facilitates detailed kinetic studies, especially those focusing on repression, which might otherwise be obscured by reporter protein accumulation.
The multiple microRNAs-one mRNA paradigm was tested with the SwitchGear Genomic GoClone system, using UCP1 as the single thermogenic target gene. In order to explore the possible interactions between various huma miRNAs and the 3′UTR region, the 5′UTR region and the promoter/enhancer region of the human UCP1 gene in Hela and HepG2 cells, three reporter constructs were made:

    
    
        1. A human UCP1 3′UTR construct containing a reporter gene driven by a strong constitutive promoter (RPL10_prom) with a 2,218 bp 3′UTR fragment of the human UCP1 sequence cloned in the 3′UTR region of the reporter gene. The effects of a specific miRNA mimic, inhibitor, or non-targeting control on this reporter's activity are compared to those of an empty—3′UTR and an Actin Beta—3′UTR to identify effects that are specific to the putative UCP1 3′UTR construct.
        2. A human UCP1 Promoter construct containing a reporter gene driven by a 4,147 bp 5′UTR fragment of the human UCP1 sequence that spans the Transcription Start Site and upstream region covering the methylation region and the enhancer region of the human UCP1 gene sequence. The effects of a specific miRNA mimic, inhibitor, or non-targeting control on this reporter's activity are compared to those of an Actin Beta Promoter to identify effects that are specific to the putative UCP1 5′UTR construct.
        3. A human UCP1 Enhancer Region construct containing a reporter gene driven by a short minimal promoter from the HSV-TK locus with a 601 bp 5′UTR fragment of the human UCP1 sequence that spans the Enhancer Region of the human UCP1 gene sequence. The effects of a specific miRNA mimic, inhibitor, or non-targeting control on this reporter's activity are compared to those of an empty 5′Enhancer Region to identify effects that are specific to the putative UCP1 5′Enhancer construct.
    
    


In addition, miRNAxxx—3′UTR constructs were made. They contain the reporter gene driven by a strong promoter (RPL10_prom) with a perfect match to the target sequence of miRNAxxx cloned into the 3′UTR region of the reporter gene. The effect of a miRNA mimic, inhibitor, or non-targeting control on this reporter's activity can be compared to EMPTY—3′UTR and Actin B—3′UTR to determine whether a miRNA mimic's or inhibitor's activity can be reasonably detected in the experimental cell type. If the cell type has no endogenous expression of the miRNA in question, the addition of a mimic should knock down the activity of this reporter, and the addition of an inhibitor should have no significant effect. If the cell type has high endogenous expression of the miRNA in question, the addition of an inhibitor should increase the activity of this reporter, and the addition of a mimic should have no significant effect. The range of endogenous miRNA expression in Hela and HepG2 cell types is broad, so the synthetic target activity changes are likely to reflect this variability.
For each miRNA candidate (38 in total), the following conditions were tested:

    
    
        miRNA mimic (specific)*8 reporter constructs in Hela cells
        miRNA mimic (specific)*8 reporter constructs in HepG2 cells
        miRNA mimic non-targeting control*8 reporter constructs in Hela cells
        miRNA mimic non-targeting control*8 reporter constructs in HepG2 cells
        miRNA inhibitor (specific)*8 reporter constructs in Hela cells
        miRNA inhibitor (specific)*8 reporter constructs in HepG2 cells
        miRNA inhibitor non-targeting control*8 reporter constructs in Hela cells
        miRNA inhibitor non-targeting control*8 reporter constructs in HepG2 cells
    
    


To the extensive list of miRNAs that may bind to the UCP1 sequence, 8 filters were applied (in addition to required binding to UCP1 3′UTR region) to reduce the number of miRNA candidates to be tested. These filters were length of binding sites, number of binding sites, binding to the 5′UTR region, chromosomal clustering with other miRNAs, intronic location, binding to the Enhancer Region, binding to the Methylation Region and proof of experimental evidence of a relation to UCP1. 38 miRNAs that met at least 3 of these criteria were tested (Table 17).







TABLE 17







miRNA with putative binding sites in the UCP1 gene sequence.




















# of 
Binding
# of






Exp. 



miRNA
criteria
length
sites 
3′UTR
5′UTR
Clustering
Intronic
Enhancer
Methylation
Evidence





















1
hsa-miR-130b-5p
7
11
3
+
+
22

+
+
+


2
hsa-miR-328
6
10
4
+
+


+
+
+


3
hsa-miR-655
6
10
5
+
+
14


+
+


4
hsa-miR-19b-2-5p
5
10
4
+
+
X



+


5
hsa-miR-26a-2-3p
5
10
7
+
+



+
+


6
hsa-miR-367-3p
5
10 to 18
3
+
+
4

+




7
hsa-miR-371a-5p
5
10 to 12
9
+
+
19



+


8
hsa-miR-377-3p
5
10 to 14
5
+
+
14



+


9
hsa-miR-378a-3p
5
7 to 13
19
+
+

+


+


10
hsa-miR-382-3p/5p
5
15
2
+
+
14






11
hsa-miR-421
5
10
5
+
+
X



+


12
hsa-miR-515 -3p
5
9
3
+
+
19

+

+


13
hsa-miR-620
5
10
7
+
+


+

+


14
hsa-miR-941/2
5
9
5
+
+
20


+



15
hsa-miR-1179
4
11
3
+
+
15






16
hsa-miR-1302
4
10
5
+
+



+



17
hsa-miR-146a
4
9 to 10
8
+
+




+


18
hsa-miR-181c
4
9
5
+
+
19



+


19
hsa-miR-203
4
9
1
+

14


+
+


20
hsa-miR-331-5p
4
8 to 15
6
+
+
12






21
hsa-miR-422a
4
7 to 14
6
+
+




+


22
hsa-miR-452
4
8
7
+
+
X



+


23
hsa-miR-491-5p
4
10
3
+
+







24
hsa-miR-501-3p
4
10
2
+
+
X



+


25
hsa-miR-543
4
10 to 14
4
+
+
14






26
hsa-miR-545
4
11
2
+
+
X



+


27
hsa-miR-549
4
13 to 14
3
+
+




+


28
hsa-miR-643
4
10 to 14
9
+
+




+


29
hsa-miR-651
4
10
6
+
+




+


30
hsa-miR-654-3p 
4
8 to 10
11
+
+
14






31
hsa-miR-21-5p
3
10 to 14
2
+
+




+


32
hsa-miR -211-5p
3
11
1
+




+
+


33
hsa-miR-22-3p
3
9
5
+
+




+


34
hsa-miR-30h-5p 
3
10
1
+

8



+


35
hsa-miR-325
3
7 to 8
11
+
+




+


36
hsa-miR-362-5p 
3
10
1
+

X



+


37
hsa-miR-504
3
9
2
+
+



+
+


38
hsa-miR-552
3
9
3
+
+




+









In these Luciferase reporter gene assay experiments, a miRNA candidate was considered to interact with UCP1 if both the specific miRNA inhibitor increases the luciferase signal and the specific miRNA mimic decreases the luciferase signal with an Inhibitor/Mimic Ratio≧1.5 and or/a p value<0.05. These selection criteria identify 9 miRNAs (miR-19b-2-5p, miR-21-5p, miR-130b-5p, miR-211, miR-325, miR-382-3p/5p, miR-543, miR-515-3p, and miR-545) (Table 18). A few more barely missed these selection criteria; they are miR-331-5p, miR-552, miR-620, and miR-1179.







TABLE 18







miRNA identified as regulators of UCP1 by luciferase reporter assay.






















# of 
Binding 
# of 





Meth-
Exp. 



Cell Line
miRNA
criteria
length
sites
3′UTR
5′UTR
Clustering
Intronic
Enhancer
ylation
Evidence






















1 
Hela
hsa-miR-130b-5p
7
11
3
+
+
22

+
+
+


2

hsa-miR-328
6
10
4
+
+


+
+
+


3

hsa-miR-655
6
10
5
+
+
14


+
+


4 
Hela + HepG2
hsa-miR-19b-2-5p
5
10
4
+
+
X



+


5

hsa-miR-26a-2-3p
5
10
7
+
+



+
+


6

hsa-miR-367-3p
5
10 to 18
3
+
+
4

+




7

hsa-miR-371a-5p
5
10 to 12
9
+
+
19



+


8

hsa-miR-377-3p
5
10 to 14
5
+
+
14



+


9

hsa-miR-378a-3p
5
7 to 13
19 
+
+

+


+


10
HepG2
hsa-miR-382-3p/5p
5
15
2
+
+
14






11

hsa-miR-421
5
10
5
+
+
X



+


12
Hela
hsa-miR-515-3p
5
9
3
+
+
19

+

+


13

hsa-miR-620
5
10
7
+
+


+

+


14

hsa-miR-941/2
5
9
5
+
+
20


+



15

hsa-miR -1179
4
11
3
+
+
15






16

hsa-miR-1302
4
10
5
+
+



+



17

hsa-miR-I46a
4
9 to 10
8
+
+




+


18

hsa-miR-181c
4
9
5
+
+
19



+


19

hsa-miR-203
4
9
1
+

14


+
+


20

hsa-miR-331-5p
4
8 to 15
6
+
+
12






21

hsa-miR-422a
4
7 to 14
6
+
+




+


22

hsa-miR-452
4
8
7
+
+
X



+


23

hsa-miR-491-5p
4
10
3
+
+







24

hsa-miR-501-3p
4
10
2
+
+
X



+


25
Hela
hsa-miR-543
4
10 to 14
4
+
+
14






26

hsa-miR-545
4
11
2
+
+
X



+


27

hsa-miR-549
4
13 to 14
3
+
+




+


28

hsa-miR-643
4
10 to 14
9
+
+




+


29

hsa-miR-651
4
10
6
+
+




+


30

hsa-miR-654-3p
4
8 to 10
11 
+
+
14






31
Hela + HepG2
hsa-miR-21-5p
3
10 to 14
2
+
+




+


32
Hela
hsa-miR-211-5p
3
11
1
+




+
+


33

hsa-miR-22-3p
3
9
5
+
+




+


34

hsa-miR-30b-5p
3
10
1
+

8



+


35
Hela + HepG2
hsa-miR-325
3
7 to 8
11 
+
+




+


36

hsa-miR -362-5p
3
10
1
+

X



+


37

hsa-miR-504
3
9
2
+
+



+
+


38

hsa-miR-552
3
9
3
+
+




+









Out of these 9 selected miRNAs, 3 appear to bind to the 3 regions of UCP1 which were studied (miR-21-5p, miR-211, and miR-515-3p); 3 appear to bind to 2 regions of UCP1 (miR-19b-2-5p, miR-130b-5p, and miR-325), and 3 bind to a single region of UCP1 (miR-331-5p, miR-543, and miR-545). All but miR-331-5p appear to bind to the 3′UTR region of UCP1 (Table 19).







TABLE 19







miRNA identified as regulators of


UCP1 by luciferase reporter assay.












miRNA
UCP1 3′ UTR
UCP1 Enhancer
UCP1 Promoter















1
mir-21-5p
X
X
X


2
miR-211
X
X
X


3
mir-515-3p
X
X
X


4
mir-19b-2-5p
X

X


5
mir-130b-5p
X
X


6
mir-325
X
X


7
miR-331-5P

X


8
mir-543
X


9
mir-545
X









Further screening is performed by transfection of the promoter/3′UTR library into human adipocytes or adipose-derived mesenchymal stem cells in cell culture, followed by addition of miRNA agents (e.g., agomirs or antagomirs) to the cell culture. Measurement of luciferase activity and identification of mRNAs is performed 24 hours after transfection and addition of miRNA agents.
In order to confirm the results of the transfection experiments set forth above over a longer time frame, lentiviral transduction experiments are performed using lentiviral vectors containing the miRNA agents of interest (from System Biosciences (SBI) collection of miRNA precursors expressed in the pMIRNA1 SBI vectors allowing the expression of the copGFP fluorescent marker). Specifically, cells containing the promoter/3′UTR library are transduced with lentiviral particles at an MOI of 1:10 and GFP-positive cells are sorted by FACS, according to the supplier's instructions. The level of expression of the mature miRNAs and their targeted mRNAs is assessed at several time points (0, 3, and 6 hr.; 1, 4, and 7 days) by Taqman Quantitative Real-time PCR in control cells (HEK293 cells), Human Adipose-Derived Mesenchymal Stem Cells, Human Subcutaneous Preadipocytes, and Human Proliferating Subcutaneous Adipocytes. Pooling of RNAs from 5 different time points after transduction is optionally employed to reduce the complexity of the qRT-PCR based screening approach while preserving the detection sensitivity.
Example 5
Proteomic Profiling
Proteomic Profiling is also used to identify novel miRNA targets involved in thermogenesis.
Shotgun proteomics is a method of identifying proteins in complex mixtures using high performance liquid chromatography (HPLC) combined with mass spectrometry (MS). Transfected and transduced cells with miRNA agents and promoter/3′UTR library (as described in Example 4) are harvested and lysed to produce crude soluble (cytosolic) and insoluble (nuclear) fractions. Peptides are from these fractions are then separated by HPLC and analyzed using nanoelectrospray-ionization tandem MS using the isotopic labeling technique SILAC to quantify protein abundance. Spectra are searched against the Ensembl release 54 human protein-coding sequence database using Sequest (Bioworks version 3.3.1, Thermo Scientific).
To avoid missing low abundance proteins, a targeted proteomics approach is also employed to accurately quantify a set of proteins that are known regulators of adipogenesis, adipocyte differentiation and BAT function. Some examples include UCP1, KDM3A, PRDM16, PPARA, PPARGC1A, CEBPB, CIDEA, BMP7, COX7A1, SIRT1, SIRT3, DIO2, FABP4, ADIPOQ. These proteins are analyzed via ELISA based or Luminex based immunoassays using commercially available antibodies.
Optionally, the protein fractions are analyzed using Multiple Reaction Monitoring-Mass Spectrometry on a proteomics platform, whereby only one protein (e.g. UCP1) of the thermogenic pathway is accurately quantified using LC-MS-MS.
Example 6
Reconciliation of the Phenotypic, Luciferase/qRT-PCR, and Proteomic Datasets
The results of the in vitro experiments set forth in Examples 3-5, herein, are reconciled. Specifically, to narrow further the initial set of microRNAs, mRNAs and target proteins and pathways to a relevant yet manageable number of targets, the experimental data is integrated with Network Searches and Analyses Packages (DAVID, Ingenuity Systems IPA and ARIADNE Pathway Studio.
Global analysis of the results of the in vitro experiments set forth in Examples 3-5, herein, is performed the Business Intelligence tool TIBCO Spotfire. This allows for a visualization of the relationships between the miRNA agents and target gene.
Example 7
Animal Models of Obesity
Several animal models of obesity have been developed and validated (Kanasaki K et al., J Biomed Biotechnol., 2011:197636 (2011); Speakman J et al., Obesity reviews: an official journal of the International Association for the Study of Obesity, 8 Suppl 1:55-61 (2007)). The most commonly used are the Leptin Signaling Defects Lepob/ob and Leprdb/db Mouse Models as well as the High-Fat Diet model in C57BL/6J mice (Wang C Y et al., Methods in molecular biology, 821:421-433 (2012). This diet-induced obesity (DIO) model closely mimics the increased availability of the high-fat/high-density foods in modern society.
A DIO mouse model is used for in vivo validation of the effectiveness of the miRNA analogs described herein for the increase in thermogenesis and/or the treatment of obesity and other metabolic disorders (Yin H et al., Cell Metab., 17(2):210-224 (2013)).
DIO mice are administered one or more of an hsa-let-7a agomir, hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-1 antagomir, hsa-miR-19b agomir, hsa-miR-19b antagomir, hsa-miR-30b agomir, and hsa-miR-30b antagomir. Rosiglitazone is used as a positive control. Food intake, blood metabolic parameters, body composition (body weight, body fat, bone mineral and lean mass, body fat distribution, body temperature, O2 consumption and CO2 production, exercise induced thermogenesis, cold induced thermogenesis and resting thermogenesis are measured in the mice prior to treatment and after treatment. A reduction in body mass or body fat or an increase in body temperature or any kind of thermogenesis indicate the in vivo effectiveness of the administered composition.
Example 8
Nucleic Acid Sequences of Human UCP1 and UCP2 Genes and Transcripts
The nucleic acid sequence of the 1,462 base pair (bp) transcript ENST00000262999 of the human UCP1 gene is as follows (Exons in capital letters) [SEQ ID NO: FROM 351-363]


























SEQ



Exon/


Start
End


ID


No
Intron
Start
End
Phase
Phase
Length
Sequence
NO:








5′





...gtcggttcaaaaaacagaaatcgg
351



upstream





gtttgctgcccggcggacaggcgtga




sequence









 


1
ENSE00001
141,489,959
141,489,758
—
0
202
AGAGCAAGGG AAAGGAACTT CCTCCACCTT CGGGGCTGGA
352



081761





GCCCTTTTCC TCTGCATCTC CAGTCTCTGA GTGAAGATGG










GGGGCCTGAC










AGCCTCGGAC GTACACCCGA CCCTGGGGGT CCAGCTCTTC










TCAGCTGGAA TAGCGGCGTG CTTGGCGGAC GTGATCACCT










TCCCGCTGGA CACGGCCAAA GTCCGGCTCC AG



 



Intron
141,489,757
141,489,132


626
gtagctaggc agaggggtaa gacaa...tgttc tgcacctttc
353



1-2





ttatttccag



 


2
ENSE00001
141,489,131
141,488,933
0
1
199
GTCCAAGGTG AATGCCCGAC GTCCAGTGTT ATTAGGTATA
354



009006





AAGGTGTCCT GGGAACAATC ACCGCTGTGG TAAAAACAGA










AGGGCGGATG AAACTCTACA GCGGGCTGCC TGCGGGGCTT










CAGCGGCAAA TCAGCTCCGC CTCTCTCAGG ATCGGCCTCT










ACGACACGGT CCAGGAGTTC CTCACCGCAG GGAAAGAAA



 



Intron
141,488,932
141,484,673


4,260
gtaagccgtg agcgttcctg ggagg...aataa ttttttttct
355



2-3





ctctggatag



 


3
ENSE00001
141,484,672
141,484,472
1
1
201
CAGCACCTAG TTTAGGAAGC AAGATTTTAG CTGGTCTAAC
356



081759





GACTGGAGGA GTGGCAGTAT TCATTGGGCA ACCCACAGAG










GTCGTGAAAG TCAGACTTCA AGCACAGCCA TCTCCACGGA










ATCAAACCTC GCTACACGGG GACTTATAAT GCGTACAGAA










TAATAGCAAC AACCGAAGGC TTGACGGGTC TTTGGAAAG



 



Intron
141,484,471
141,484,366


106
gtaactaact tcaaaatggg tttta...acatt ttcttttttt
357



3-4





ttttccccag



 


4
ENSE00001
141,484,365
141,484,264
1
1
102
GGACTACTCC CAATCTGATG AGAAGTGTCA TCATCAATTG
358



081762





TACAGAGCTA GTAACATATG ATCTAATGAA GGAGGCCTTT










GTGAAAAACA ACATATTAGC AG



 



Intron
141,484,263
141,483,528


736
gtaacttccc atttcatata acaaa...gacc tgtttcatcg g
359



4-5





atccatttta



 


5
ENSE00001
141,483,527
141,483,347
1
2
181
ATGACGTCCC CTGCCACTTG GTGTCGGCTC TTATCGCTGG
360



081763





ATTTTGCGCA










ACAGCTATGT CCTCCCCGGT GGATGTAGTA AAAACCAGAT










TTATTAATTC TCCACCAGGA CAGTACAAAA GTGTGCCCAA










CTGTGCAATG AAAGTGTTCA CTAACGAAGG ACCAACGGCT










TTCTTCAAGG G



 



Intron 
141,483,346
141,481,165


2,182
gtaagatatg atcttgtgta tctgt...cgaac gatgacatgc
361



5-6





acttttctag



 


6
ENSE00001
141,481,164
141,480,586
2
—
577
GTTGGTACCT TCCTTCTTGC GACTTGGATC CTGGAACGTC
362



081760





ATTATGTTTG










TGTGCTTTGA ACAACTGAAA CGAGAACTGT CAAAGTCAAG










GCAGACTATG GACTGTGCCA CATAATCAGC TTCAAGAAAA










TGATGTAACA TACCAGTGGG AATCTTGCTG ACTGGATCAT










AAAAACAAAC AAAACTTATT CACTTATTTT AACCTAAAAA










GATAAAGGAA TTTTGGCAGA GAATTTTGGA CTTTTTTATA










TAAAAAAGAG GAAAATTAAT GCCTATTTCA TATAACTTTT










TTTTTTTCTC










AGTGTCTTAA GAAGGGGAAA GCAAAACATT CAGCATATAC










CCTGGCAAAT GTAATGCAGA TAAGCTACTG CATTTGACCA










TTTCTGGAGT GCAATTGTGT GAATGAATGT GAAGAACTTT










AACATGTTTT AATTACAATT CCAACTGGTG GAAAAGAAAC










TGAGTGAAAT GCAGTTTATA TTTATAAATA CTTAAAAATG










AAGTTATTAA AAATATTAGT TTTTATTAAC CACAGTTGTC










AGTTAATATA TTCAATAAAA GTATTGCTAA TACCTTTT



 



3′





aaagtttgtcttttgagatctatacct
363



down-





gggtgtaagagtcaagttcacta...




stream










sequence









The nucleic acid sequence of the 9,371 base pair (bp) of the human UCP1 gene (ENSG00000109424) is as follows (Exons are highlighted) [SEQ ID NO: 364]:








>chromosome: GRCh37:4:141479988:141490559:−1



AGAGAAGGCC GCAAGGTGCC TGCAAGATGT CTGGGGAGTT GGAGGAATGG AAGAGTGCCC      60


 


CGCTCTTCCT TCTGGGAGAG CTCCAGCTAG GCAGAACCTT TCACCAAGGC TCTGATATCG     120


 


TGCTGGTTTC CGAAAGCCCC AGCCGAAGGT GTGCAGCCAA AGGGTGACAG AAGGTGAGGC     180


 


ACGTGCGGGG GCGCGGGTGC TGACCGCCGC GGTGCGCCCT CCCTCCGACG TGCGGTGTGC     240


 


GGGGCGCAGA CAACCAGCGG CCGGCCCAGG GCTTTCGGGG AGCGAAGCAG GGCTCCCGAG     300


 


GCACCGAGCG AGAATGGGAA TGGGAGGGAC CCGGTGCTCC CGGACACGCC CCCGGCAGGT     360


 


CCCACGCCCG GGTCTTCTGA GACCTCGCGC GGCCCAGCCC GGGAGCGGCC CAGCTATATA     420


 


AGTCCCAGCG GAAGACCGGA ACGCAGAGGG TCCTGCTGGC GCGAGGGTGG GTAGGAGGGG     480


 


ACGCGGGGAC TCGGCCCCCA ACACCGCGCT CCGTCTGCAG CCGCCGCCTC TGCACCGCCG     540


 


CTGCCCGGCG GTCGGTTCAA AAAACAGAAA TCGGGTTTGC TGCCCGGCGG ACAGGCGTGA     600


 


























AGAGGGGACG CTGTTGCGTG CATTCCATTT ATTCTCTGCT TTGGTGTAAC CACTGTTTCT     900


 


AGGTAGGGTA GGTGACCTTC CAAAGCAGTC TGGCCTTGTC CCAGGGCTGG TGCTTTAGGA     960


 


TGGGAAACTG GAACTTTTTC TGGGATTAGC TGAAGAACCA CCAGGGCCAC AGAGAATGGG    1020


 


TTGACCATGA CTACTACCAA ATTCTCCCAA AATTTAGGGT GCACTTAGTA TTTTAAGAGC    1080


 


TGAGAATATT GGCCTCTCCT GAGTTTACTA GTCAGGTGCT TTTTCCTTTC TTTGATTCTT    1140


 


CGGGGGTTCT GTCCTATCCT ACTGCCCTAG GGGTTCTGGA GAGTTCCTGG GGAGGGGGAT    1200


 


ATTCAAAATG TGCATTGTAG CCAGCCTCCC TCCATCTGCG CGTGAGCGAA CACACACACA    1260


 


CACACACACA CACACACACA CACACACACA CACACACGGT AGAGGGAGGT GGATGGAAGA    1320


 


GGAATGTTGC TGAGAAAAGA AACGGAAAAT AGGAACACAG GGGGAAATCT TGGCTTAAGA    1380


 
































TCCAAAAAGT GTAACACACA GAGGAGTGGT TTTCATAACA AATTGGCGAG AAAACATTCA    1740


 


TATTTGAACT CTCCCTTCCC CAAACATTAG CTCATTGTTC ATAGAAAAAA GTATGCAAAA    1800


 


TCGATTTTTT AGATGCAGAT ATATACTTGT AAAGGTCACC CAGTCATGGA AGTTTTGTGC    1860


 


CCAGTTTGGA TCTCCATCTG GAGAATATGG GTGGGCTACA GAAAAATGTT TAACTTAAAG    1920


 


TTCTCCAAAG AGGGAAGTAT ATCAGAAACA TCTATGGAGC TTGTCAGAAA TCCAAACGAG    1980


 


GACTACCATG GTCCTCTGAG TCTGAATCCT CAGGCTAGAG ACCAGAGTGT CTTTCCACAA    2040


 


GCTTCCCTCA TCATTTGTGT ATGCAACAAA GTTCAAAGCC TTCTGTTTGA AGCAAAGAAA    2100


 


GCCAGACTTT GTGAAGAGAG TTGAAAGGAC AGGAAAAGAC ATATTTCCTC TTAAGAGGTT    2160


 


CCTCATCAGG TCCAGGAAAG ACCAGAGCAG AAAAAGTGGA CGAATGCTGC AGGGAGTTTG    2220


 


TTTAGGGGAA AAAGAAAAGG AAACATATTT CCTGAGTGCC AGTGCACTCT AAGAATTCCT    2280


 


GTCACTTTAG GTAGCATTTA TTTGAGGGCT TAACTATGAA CCAGACATTG TTCTAAGTGC    2340


 


TTCAGATACA TTATAACTGG AAGGGTATTA GTACCATTAT CCCTTGGCAG ATGGGAAAAC    2400


 


TGAACACAGA GCAGATTCAT CACTTGCCCA AGGTCACACA GCTGGGAGGG GGCAGAGCCA    2460


 


GGGTTCAAAC CCAGGCAGTC TGGCCTCGGA CTCCAGGCTC CTAACCCTGT TCTCTACTGC    2520


 


CTTCTGCACT TCTCATATGA TTCTGCCCAT CATTCAAACC GCACAACACT GCTGTGAGTA    2580


 


AAAAGTGTTA GCCGAATATC AGGGTAGTTA AGTAACATGC ACAAAATCAC ACAGCTAATC    2640


 


AACATCAGAG GCACTTTCAT GTGGAGTAGA CAAGCCAGAG AGAAGATGTG CTGATGGCAC    2700


 


AATGAATACA TTAAGTGAAA TCCACCTTGT AGATTTCATC ATTTCTGCTG TGAGTAACCT    2760


 


TCAATACTAT AATTTTATGG GATAATTTAT AAATGTTGTC TATACAAATA TATAAGTTAT    2820


 


ACTTATCCAC ACAAGTACTT TCAAAGTGAA GATAAAGTCT GGATGTTACT AGATCAAAAC    2880


 


TGCATTTTTT TATTTATAGA TGTAGCAAGA GAGGAAACAC AAAGGAGGTA AAGCTGCCCG    2940


 


TTCAGGTGGT TTTCTTCACA GATTGACTGT TCTACCAATT GTTGTGGACT TTGGGCACCA    3000


 


AATTAATAGG ATATATGTTG GCAGTGTTCT ATGTTATATA GATTCAGTTT ATTTAGTAGG    3060


 


CTTTATTGAA CTGCCATGTG CCAGTAACTA TGTTAGATGT TTAGATGGCA GATGTGTCTC    3120


 


TAGACAGAGC TTACAGTTGA GAGTATGGGT TGTGTGGGGA GAAGTGAATA GATGACTATA    3180


 


TTCCATGATA CATGCTGTAT TACAATACAG TCCTACTTCA CTTAACGATG GGGATACATT    3240


 


CTCAGAAATG AGTTAGGAGG CAAATTGGTT GTTGAATGAA CATCACAGAG AGCACTTACA    3300


 


CAAACCTAGA TGGCATAGCC ACACCTAGGC TATATGGTAT AATCTATTGC TCCTAGGCTA    3360


 


CAAACCTGTG CAGCATGTTG GTATTGAATA CTACAGGCAA TTGTTACATA AAGTTAAGTG    3420


 


TTTGTGTACC TAAAAATAGA AAAGGTAATG CATTACACTA CAGTCTTATG GGGCTGGGAT    3480


 


GTCACTAGGT GATAGGAATT TTTCAGCTCT GTTCTAATCT TACGGGACCA CCATCATGTA    3540


 


TGCAGCACAT GACTAACTGT AATTACAAGA TGGTGGCTAT ATTAAACAGA ACTACTTAAG    3600


 


CTAGCCATGG AGGTATGGTC CGTGAGATTT TCCTGAAGAA TTAACGTCTG GATCAATTCT    3660


 


GGAAGGGCCA GCAGGAGTAC TCCAGGCAAA GGGGTGAGAA AGGAGCTTCC AAGTAGAGTG    3720


 


AAGGTCATGT GCAAAGACTC AGTGAGGAGT CGAGTGAACA TAGCACAGGG AGGACATGTT    3780


 


GGTGAGGAAG GAGGGGTGAA GCCACAGAGA CAGGAGGGAG CCAGATGACA GAAGGCCTTG    3840


 


CAGGCGGTGC TAAGGAGTTT GGATTTTATC CTTACAGTGG TGGGAAGTCA TTGTAAAAAT    3900


 


ATTAAGCAAG GGAGTGGCAT AAACAATTTA CATTTTCAAA AGATCACTTT GGCAGCAGAT    3960


 


AGAGTATATA TGTAAAAGGA GTAAGAAAGA GGTAAGTTAG AAAGCAAGAA ATGATCAGGG    4020


 


TATGCCCTAA AACACTGGCA ATAGGGAAAA AGAGATGTCA ATCAGAAAGA TTGAGAAAGT    4080


 


ATAATTGAAT TGACTTGGTG AACAAATAGA AGTAAGGCAT AAGGGACAGG TAGAAATATG    4140


 


AGATGACTTC CAAGTTTCTG TTTAAAGATA CCCTTTATTG AGAGAGGATG TATAGAAGCT    4200


 


GTCTTAGGGG GAAGACAAGA AATTTGGTTT AGGCCATGTC AACAGGTAAT GGCCAGTAGG    4260


 


CACATGATTC AGTTTATTTA GTGGGCTCCT TTTAGGAGAA AATCTGAGCC AGATTCCAGG    4320


 


AAGTCACAGC AGGGACTACC AATAGGGTCA AACAGCAGAG AGTGTGGAAA GGACTGAAAA    4380


 


GTGATCATTG TACATAACAA ATAGAAGCTC ACTGATTTTC TAGCAAAAAC ATCTTCAGCA    4440


 


GAGTAGCGTG GTATAAGCTA TATTGTAGGG GACTGAGGAA GAAATGGGCT CTGAGAAGTA    4500


 


AAGACAAACA ATATGTTTTG TAAATAAATT TCTTTTAGTT CTTAAAAAAA AAGCCTCTTT    4560


 


TCCAGCTTGA TTGGGAAGTG AAGAGAGGGA TTTGAAAGTT GGAGATTGGA GGATAGGATG    4620


 


AGTACATCAA GATACACTAC GTTGTAGTGC AGTGCATTAC AAATGTGAGC TAAAAGTGAA    4680


 


GGCATTTGTA ATCATATGAT ATTGCTAATT AAAAGACAGC TGTCAGTCAT ATGCCCAGCT    4740


 


CCTGGTAAAG CATGATGAGA AGAGTACAAT CATGGTAGTG ATTTAAAAAT TGCTGCCAGT    4800


 


TTTGTGGATT TTCTTTATGC TAGACAGTGT AAGCTCTTTA TCAATATTAT TTAACTCACA    4860


 


CAACTCTAAG AGGTAGATAT TATTATCCCT TTTTGACAAA TTAGGAAACA GAATTATAAT    4920


 


GACTGAGAAA GTCTCTGCTG AGTAAATGTT ACTGAACCTT AATTTTATGT TTACTTAATG    4980


 


ATAGAAATGA ATATTGGGCT TCAAGACTAT TTGTACTTAA TGAAATCTGT CTTGAGCAAC    5040


 


ATAAGCTATT TTTTTCAAAA TTTTAAGACA AAAATCACTT TCTTCTCTCC TGTCTTCTTA    5100


 


TTTTTGTTCC CTTCACATGT TGTAGCCTAA CACTACTTGA TGGCCCATTT TGGTGCAGTT    5160


 


TGTCCACTGG GCTTCATCTA AGGCCACCAA GTCCCATAAT TAACATGATC ATTCGTGGGA    5220


 


GAAAGATCAA GCCTCATTGG TGATGGGTGC CTCCTCACAG TCGGATAATA CTGAAAAGAG    5280


 


AGCTAAATGT GGGAAAGAAC CAAGTTGAAC ACAGGAAAGA ATCAGGCCAC TGTGAAAATA    5340


 


AGCATTGTGT TTTCTTGTTC CTTGAAAGTC TTCATTTTTA AAAAATTTCA GACACCTGAA    5400


 


GTTTTCTAGC CTTACTCTGA GTTGACGCAC ATTTAGTACA TGATCAACAC ATAAACAAGC    5460


 


ATTAGAGAAA TAGAAAAGCT GTAAGAATAC AAAAATATGG GCCAGGTGGG TGGCTCATAC    5520


 


CTGTAATCCT AGCACTTTGG GAGGCCGAGG CAGACGGATC ACCTGAGGTC AGGAGTTCAA    5580


 


GACTAGCCTG GCCAATATAG TGAAACCCTG TCTCTACTAA AAATACAAAA CTTAGCAGGC    5640


 


TGTGGTGGCA CGTGCCTATA ATCCCAGCTA CTTGGGAGGC TGAGGCAGGA GAATCTCTTG    5700


 


AACCCGGGAG GCGGAGATTG CAGTGAGCCA AGATCACACC ACTGCACTCT AGCCTAGATA    5760


 


ACAGAGCAAG ACTCCATCTC AAATACAAAA AAAATACAAA AATATGAACC ACTGAAAATT    5820


 


AAAAAGACAT GCATGCATTC TAGGTCTTTA ATTTTTTTTC TTAATAATTT TTTTTCTCTC    5880


 


























CAAAGCACAT ACATACAACT AGCAACTTAT TGTAAAGTAG AGTTAATAAA CATTTTCTTT    6180


 














CTTCCCATTT CATATAACAA ACAGGTCTGC ACCTTTAGAA GTTCATCTTG GAGCTTCTGC    6360


 


AGCCACCTTA TACTCAATCT CTTAACTCCA ATAGTTTTCT CTTTTTAAAA ATTAAGTAAT    6420


 


TTTGAACCAT ATATAACTTT GTGAGAAGCA GGAAAAGACC AAAATATTAA GTTTAAGAAG    6480


 


TTTTGCCACA ACAAAAATAT TTTGCAACAA AAATAACAGG CAATTTCATG TCAGCATTAT    6540


 


TCTCATTTAA TACTAATATA TGGGACTTTT GTTAGAATCT TATTCTTTAT ACAGCAGAAT    6600


 


TCAGGAGGTA AGTCCATCCT GCATACTATA TCCAAAAGAT CTAGTTATAA AAGGAGCTTA    6660


 


TCAGTGGTCT CATCCAAAAA GTAATACCAT AAGATAGGTT CTTAAAAATA ATATTCTAAC    6720


 


AACTTCTAGA GACATTGAAA TTTCCCTTAT TTCAATAAAA AAGTATTAGA TGCTCATATA    6780


 


TTAGGCATTA TTACAGGCCT TAAAGGCACA GAGGAAACTA ACAGTTTACT TTCCTAAAGT    6840


 


GTTAACAATC TATTAAGCCA TTTACTCTTT ACCTTCTTTT TCTAGTGCAA TACCTTTCTT    6900


 


ATTTTATTTT ATTTATTTAT AAGACATCTT CATTGACCTA CTGTTATCAA TAGGTTTATA    6960


 


AAGATATGAC AGATAACTAA ATTGCAAGCC CCCAAAAGTC TGATGTTGAC CTGTTTCATC    7020


 


























GCTTTGGGAC ACTCTCATGT CAAGCAACCG ACATTTAGCT TACAAGCCTT AGTATATTCA    7320


 


TATACTTAGT ATTGACTTTT CCTTGCCACA GATTTCTCCA ATCCACCAAT TCCACTGTGC    7380


 


CAGAAAGTAA AAAGCCATGA TATTCAAATT TTCTCAACTT TGATCAAAGG CTCATTCAAG    7440


 


ACCAGTGCCT TTTCCACTGG TCCCAATCTA CTGGAAATGC AGACAGTATT TTGCCTTCTC    7500


 


TGGGCAAGAA AGTTATAAAG TAGAGGGAAA TCATAATAGA GAGCTATGAG AGAACAAGAT    7560


 


TTGATTTGAT TTAATTTGAT GGACTCAAGT TTTAACATTG TAAAACTAGA GATAAGACAT    7620


 


CACCACCAAT CTAGAAAAGT GATGCAGAAA AGTATTTGAT TTGGGTAATT ATTACACTCA    7680


 


CCTAGAAACA AGTGTTGTGT AATAGATTAC ATATTTCCAT AATGCAATGT TGTATCAGAA    7740


 


ACTACCTTCC TAAGAAAATA TAGTATGGGC TCGGCGTGGT GGCTCGCACC TGTAATCCCA    7800


 


GCACTTTGGG AGATGGAGGC AGGAGGATCA CTTGAGCCCA GACTGGGCAA CAAAGCGAGA    7860


 


CCCTGTCTCA ACAAAAAATT TAAAAATTAG CTGAGTGTGG TGGCACGCAC TGATGGTCCC    7920


 


CTCTACTTGG GAAGCTGAGG CAAGAGGATC TCCTGAGCCC AGGAGTTCAA GGTTTCAGCG    7980


 


AGCTATGATT GTGCCACTGC ACTCCAGCCT GGGAGACAGA GCAAGTCCCT GTCTCAAAAA    8040


 


AGAAGAAGGA GAAGGAGGAG AAAATACAGT ATTAAGTAAT CTGTCAATAT ATTCCACAAG    8100


 


GATTACACTA GTGGTTTAAT AATAAAATTA TATTACCTTT TTAAATTGTA AGGCCATTCC    8160


 


TCAAGCTTTA TAAATTAAGC ATGAATGCAT CATACACATT TTATAAAAAG TTCCAACTCA    8220


 


TCATAATCTG TACTTATGAT ACATTAATAC AAATGAAGTT CATTATAAAA TTAACTTAAA    8280


 


ATGGATATAC CAGTTATTAA ACCATTAACC ATTTAATAAT TTTATTTTTT TCAAATTTAA    8340


 


AAACCTTTTG GGGAAGAAAT ACTACAACAT GGATGAACCT TGAAAACGTT ATGCTAAGTG    8400


 


AAATAAGCCA GACACAAAAG GACAAATACT GTATGATTAC ACTTAAATGA GGTACCTAGA    8460


 


GTAGTCAAAT TCATAGAGAC AGAAAGAATA GAAGTTACCA GGGGCTGGAG GTAGGAAAAA    8520


 


ATGGAGAGCT GTTTAATGGG TAGAGAGTTT CTTTTTGGGG TGACAAAAAG GTTCTAGAGA    8580


 


TGGATAGTGG TGATGGTTAC ACACAATGTG TGTGTACTTA ATGCTACTGA AATGTAATTT    8640


 


TATGATTTTT TTTTTTTGCA GCAAAATACC CCACATTGGG AAGTGAAGAG AAACATGTTA    8700


 


AGAGACTTGA AGGAAAAAAA TTGGGGCAGA GGGGTGTTTT TTATAGGTTA AACAATAAAA    8760


 


GCCATTTAAA CAGTAACAAT TTCTCTAAGG ACAAGAATCG TCAAGATTGA GACAGCACTG    8820


 


ATTTCTTGAC TCTACTCAAT ACTTCTTTGG TTTCTCTTCT TCCTTCCCCC TTCTAATAGT    8880


 


TTCCTACCTC CCATTCAGAA AGCAAAGCAA AACAAGCAAA AATTCCCCCT TCCCTCAAAA    8940


 


AAGGAAAGAG TTTTTGAAAA AGTTCATGTC AGTGAAGAAA AGACATGTTT TGGGAGTGAA    9000


 


GGATATTTGT GGATTTGTAT AGATGTGATC ATCAGGGCTG TGTTGTTTTG AAGTAATATA    9060


 


GGACATCTAG AGGAAAATTT ATTTTCAGCA GAGGAGGGAA AGATGAAGAG TAGGTACTTT    9120


 


TAAGCATCTT CACTTGAGGA GTGGCAAAAT GAGAAGCATA ACCTGCTATA ATCACTTTAA    9180


 


GAATTTCAGG CTGAGTGTGG TGGTGCAGTC TCTAGTCCCA GTTACTCCAG GAGGCTCAGG    9240


 


TGGGAGGATC ACTTAAGCCC AGGAGCTCGA GGTTGCAGTG AGCTATGATT ACACTACTGC    9300


 


ATTCCAGCCT GGGCGGCAGG GTGAAGCCTC ATCTCAAAAA TTAAAAAAAA AAAAAATCAA    9360


 




































































TAGAATACAA GACTGCCCAA TAGCAAATGC AGGTCTTTAG AATCATAGGC ATGAACCTAC   10080


 


TCTGAATGTT ATTAGTATAG ATTTTTAATG TTTAGAGTCC AGATTTGATG ACATCTCTAA   10140


 


CAACTTCTAA TCTAAGACAC TATATTCATT TTGGCAGGAT TGCTACTAGA GTCTTGGTAT   10200


 


CTGTGCTAGC ATCACATAAT TTTAGAGCTG GAGGGTACTT CTGGGAAGAC AGAGGAACAG   10260


 


TTTGAGATTC CTACTGAGAT GAAAACGAAT CTTCATGGAA TCTTTCAGCA AAGCCAAATT   10320


 


CAAATTCATC ATTAGCACCT GTAGTAACCT TTTCAATGCC TACAAACTGC ATGCAGAAGA   10380


 


GATAGGGAAA CAGTAAAACA GATATTAAAA GAAGTTTTTA AGACAAAGCC CAGCCTGATT   10440


 


TTAAGCTAAA TCCAAGGATT GGCAGCTTGG ATGAGCAGGA AGGTTACAGG CTGCCAGACA   10500


 


TCATTCTAGT TCTGTTTTAA TCAACTCCAT GTTACATTTA CTATCAGGGA TTCTCACCTC   10560


 


ACCCTCATGC AT                                                       10572






The nucleic acid sequence of the 15,901 base pair (bp) human UCP1 sequence (gi|237858805|ref|NG—012139.1| Homo sapiens uncoupling protein 1 (mitochondrial, proton carrier) (UCP1), RefSeqGene on chromosome 4) is as follows [SEQ ID NO: 365]:









CTGTACAGCT CTCCGACAAT CCCACATCTA GATGCCAAGC TGAGGTTGGC ATTCTCACTA
60



 


ATTTGCTGTT ATAAATATTA AGCTATCATA AGCGTTAGCC TACATATGAC TCTTTCATAT
120


 


GTTAGTTAAT TATTTTAGGG TAGAAATCCA AAAGTGGAGT TACCAGAAGT GGATATAGAC
180


 


ATTCTGGCTG GGTGTGATGG TTCATGCCTG TAATCCCAGC ACTTTGGGAG GCAGAGGCAG
240


 


GCGGATCACT TGAGGCCAGG AGTTTGAGAT CAGCCTGGGC CAACACAGCG AAACCCCATC
300


 


TCTACTAAAA ATTCCAAAAC TAGCCAGGCA TAGTGGCACA TGCCTGTACT CCCAGCTACT
360


 


TGGGAGGCTA AGACACAAGA ATCGCTTGAA CCCGGGAGGG AGGTGGAGGT TGCGGTGAGC
420


 


TGAGATTGTG CCACCGTACT CCAGCCTGGG TGACACAGCT AGACTCTGTT TCAAAAAAAA
480


 


AAAGAAAAAG AAAAGAAAAA AATAGACTTT CTCTTGGCTC AGTGTATACT GCCAAATTGT
540


 


TTTCCAAAAA AATTGTGTCA ATGTATAACA CCATCACTAA TATAGTATTG ATATTATGGT
600


 


TATTACATTT TAAAATTCAT AATTTGTAAT TATAACATTC ATAATTTATT ACTATTTATA
660


 


ATATTAATGT AAATGTATAT TATATATAAA TGTTATAGTA ATTATAACTT TGGTAGTGAC
720


 


AAAGTATTAA TTTATTAGGT GAAGTATATG CTTTTTTATT AGTGATAATA AATATATCCT
780


 


CTCTCCCATT ATAAAAGTTT GTATTTCTTC TTTTAGAAAT TGATTCTTCT GTCATTTGCA
840


 


CATTTATCTG TATAATTATA ACAGGGTATT TCCCAGTGGT GGCTAATGAG AGAATTATGG
900


 


GAAAGTATAG AACACTATTC AAATGCAAAG CACTGTATGA TTTTTATTTA ATAGGAAGAC
960


 


ATTTTGTGCA GCGATTTCTG ATTGACCACA GTTTGATCAA GTGCATTTGT TAATGTGTTC
1020


 


TACATTTTCA AAAAGGAAAG GAGAATTTGT TACATTCAGA ACTTGCTGCC ACTCCTTTGC
1080


 


TACGTCATAA AGGGTCAGTT GCCCTTGCTC ATACTGACCT ATTCTTTACC TCTCTGCTTC
1140


 


TTCTTTGTGC CAGAAGAGTA GAAATCTGAC CCTTTGGGGA TACCACCCTC TCCCCTACTG
1200


 


CTCTCTCCAA CCTGAGGCAA ACTTTCTCCT ACTTCCCAGA GCCTGTCAGA AGTGGTGAAG
1260


 


CCAGCCTGCT CCTTGGAATC CAGAACTACT TTCAGAATCT TGAACTTCTG TGACCTCTCA
1320


 


GGGTCCCCTT GTGTGAAGTT TTTGACGTCA GCTTCTCCTG TGACCCTTAG AAGTCACTCT
1380


 


TGTGTCTAGC ACATCCCAGG TGCTCAGTCA CCATTGAACT ACAGTCATAC TATCTCCTGG
1440


 


CAAAGGCTCT TAACTGTCCA TGTTAGCCTG ATATTAATAT CCTGGAAGCT TATACTGTCG
1500


 


TTCTTCCTTC CAGGTTTAAA TAAGGCAGCC CCTTTATCCT GTCACAGGTC CTCTCTCCCT
1560


 


ACCTATCCTT ACCTGTTTTG GATAACAACC TTTCTTATTT CTAATAGATT TATTTATTTC
1620


 


TCACATTTCC TTCCCTTATC ATAGTTTTCC TCTCACTTTC TCCTCTAGTT TGTCATACTC
1680


 


TGGCTTTAAA ACATGCAAAC ATGTGCCTTA TGGGGAAAAA AAGACAATTT TAATTTACCT
1740


 


TGCTTCTTCT TTACAAATGT ATTGTGGCTT CTTCTTATAG TCCAAATCTA AAACTCTTTA
1800


 


CCCACCCACT GCCTTGAACT CCTTCCTCGT TGTGAAAGTA GGATGGGGCA AAGAGAGAAT
1860


 


GCATGCCCCT CCCAACTGCT CAAACAAGTA AAGGTGCTGT TACAGTTATC TTTTGCTACC
1920


 


TTAATACAAT AATTATTTTA TTATATCTCA CAATTTTATG GATCAGGAAT TTAGACTGGG
1980


 


CTCAGCTAGG CGATTCTTCT GCTTTACTGA CATCATAGGA GATCACTTGG TGGTATTCAA
2040


 


CTGTCAGGTA GGCTTATCTG GAGGGTCCAA GATAGCTGTA CTCTGGTGCC TGGTGCCTTG
2100


 


GTAAAGAGGG ATGATGATGT GGGGCCTCTC CAGCATGAAC AGCCTCAGAG AAGTTTGCTT
2160


 


TCTTACATGC TGGCCCAGGG CTCCAAGAGC AAATGTTGCA GTGAGTAAAG CAGAAGATAC
2220


 


AAGGACTTTT ATAATCTGGT CTCAGAAGCC ACATGGCATC AGTTCTGTAT TATTCTATTG
2280


 


GTCAAAACAT TCATAAGCCT GCCAGATGCA AGGGGAAGGC ATATGTACCC TCATCTTTTG
2340


 


ATGGGAGGAA TGTGATGGAT TTGCAATTAT GTTTTAAAAC TACTACAGAC AGAACCACTG
2400


 


AGAAAGATTC ATGGGTAGCT TTGGGGTGAG GACTGGGAAT TAACCTGTTG ATAGCAGAGG
2460


 


TTCACTAGAG TCAACAAGGA ATAAGGTCTC CTCTTGTACA CTTTAGTCAT ACTATACCAA
2520


 


CATTCTTAAC CACTGCTTAG CCATCAGCCT CACAACATAA CAACTCCATC ATAGTTGTAC
2580


 


TCCCTAAGAT CACCAACAAT GTTAGAGTCA AATCCGGTAG GTTTTTCTTT GTTTTTGTCC
2640


 


TCCTGACATT TTTTCTAAAC TTGACACTGG TCAGACCCAA TCTTTCTTTA ATCATATTCT
2700


 


TAAATACCAG TTCTATCACT GGATATGTTA CTGTTTCTTG TTCTCACTCT ACCTTTGACA
2760


 


AAGCCATTCT TTCCAGACTA TAACTCTGGG TCTGGGTCCC CCTATGGTTT GGCCCTTGAA
2820


 


TTCTTTTCCT AGTCCTATTT GACTAGCCCC ATTTTCCCGT GAAAAGCATG CCCCTTTCAT
2880


 


TGCATCCATA TCATGACTAC CAAATACCTC CTCTATTTCT TCCTCTTTTA GCATGTTAAA
2940


 


TGCAGCTTCC TAAGCTCTCT ATCTGGATAT CAACAGTATT CTCTCCAAAT AATTCTAAGA
3000


 


CTTTAAAAAT TGGTTTAATC TTCTTACCCC TAAAATCACC CCCCTTACCA ACTGCCTCAT
3060


 


GACAATCATT GGTACTGTCA CTGAGCTTGC AACCCATGTT CTTAAACATA GAGTAATCTT
3120


 


TGACTCCACA TCTAATCATT CATAAAGCTG TATTGTCTAT CAAATTAAAT CTGACATTTA
3180


 


TGTGAGAGCA CTTCATAGTC TGTAAAGCAC TACACAGGTG ATAACATGAA GCTACACTCA
3240


 


TAATGGATTT GCAGGCTCTG CTTCTCATTT GGCTTCTACA GCCTCATCCC TCACCAACTT
3300


 


CTTGCCCTAC CTCTCTCTTT CTTCCCCATC ACCCAATTTC CCAGTCAGTC AGGCCAACAG
3360


 


AATGCATTCT ATATACGCGA CTTGCTTTCC CCAACATCTT TGCCTGTATG CATGCCACTT
3420


 


ATTTGCCTCA GTTGATCTTT ATTTCAACAA GTGTTTGCAG AGGAGAAACC TCGCTGGCTC
3480


 


CTTCTCCTTT CTATTTTTTT TCAGAGGCTA CCCGTCAGGT CAACATTGCC TTTTTCAGGG
3540


 


AAGCTCTGCA AGCCTGACCT CCCTTGGAAG TGCCTTAGGA CTGGCTTCTT GCACAGTACA
3600


 


CAACCTTTAC TTATAGAGGG TTTGGAGATT ATTCTTTATT CATGTCTTAT TTCTCCTGCT
3660


 


CCTGGAGGAG ATGACTCTGA CTTCCACTGA CTCTTTTGGG GGGCTTAAGT CAGGGTTGAG
3720


 


TACCAGAGGC CCTAAATAGC TGGACGTGGA TTCTGGTAAT ATCAAATCCA TCTTTGGCTT
3780


 


AACTGAGAGG TTCTGAAAGC TGGGACCTGA CCTTGTCCAT TTCCCTCTTT CTCCAGTTTC
3840


 


CTATTATTTC CCACTGTTTT TTTTAAAAGT TTTTTGTTTT CTTAAGTTTT CACAAGAATA
3900


 


AACATTGAAA ATAAAATTTG CACAAAGATC GAACTAGGAA AGGCCACACA ACCAACACAT
3960


 


ATTACATCAT TATAGGTAAG TTAGCAGGGA GATTTCAGAC CTGGGCTAGC TCTGGAACCA
4020


 


CATTTTACAC TGTTGAAAAT AAAAGCTGGA GTACAGATGA CTTTCCCAGG TTCACAGAGT
4080


 


TGGTAAGCTG GAGAGCTGCA CCTGGAGCCA AGCAACCTGC CCTGTCCTTT CCACTGCACC
4140


 


CTCTAAGAAA TCTAATTAGA AGGAACAGGT GGTATCTCAT TTTGTACGGT GCTTTAGCAA
4200


 


TGTACTATTT GCTTTCTAGT GTGTCTATTG TCTCGTTTGA CATCTTCTCT CAAAAAGTGA
4260


 


TGAAACGAAA CGCTCTTTTT GACAAGTTCA GAGTGCTCTT GGTTCCTGTG TGGGATTCTT
4320


 


CCAAGTCTGA ATTTGGTAGT GGGAAGAGAA GGAATCCGGA GGAAGGAGGA TGAGAAGTTT
4380


 


AAAGGAGAGG AAAGGGAAGC AGAGAAGGCC GCAAGGTGCC TGCAAGATGT CTGGGGAGTT
4440


 


GGAGGAATGG AAGAGTGCCC CGCTCTTCCT TCTGGGAGAG CTCCAGCTAG GCAGAACCTT
4500


 


TCACCAAGGC TCTGATATCG TGCTGGTTTC CGAAAGCCCC AGCCGAAGGT GTGCAGCCAA
4560


 


AGGGTGACAG AAGGTGAGGC ACGTGCGGGG GCGCGGGTGC TGACCGCCGC GGTGCGCCCT
4620


 


CCCTCCGACG TGCGGTGTGC GGGGCGCAGA CAACCAGCGG CCGGCCCAGG GCTTTCGGGG
4680


 


AGCGAAGCAG GGCTCCCGAG GCACCGAGCG AGAATGGGAA TGGGAGGGAC CCGGTGCTCC
4740


 


CGGACACGCC CCCGGCAGGT CCCACGCCCG GGTCTTCTGA GACCTCGCGC GGCCCAGCCC
4800


 


GGGAGCGGCC CAGCTATATA AGTCCCAGCG GAAGACCGGA ACGCAGAGGG TCCTGCTGGC
4860


 


GCGAGGGTGG GTAGGAGGGG ACGCGGGGAC TCGGCCCCCA ACACCGCGCT CCGTCTGCAG
4920


 


CCGCCGCCTC TGCACCGCCG CTGCCCGGCG GTCGGTTCAA AAAACAGAAA TCGGGTTTGC
4980


 


TGCCCGGCGG ACAGGCGTGA AGAGCAAGGG AAAGGAACTT CCTCCACCTT CGGGGCTGGA
5040


 


GCCCTTTTCC TCTGCATCTC CAGTCTCTGA GTGAAGATGG GGGGCCTGAC AGCCTCGGAC
5100


 


GTACACCCGA CCCTGGGGGT CCAGCTCTTC TCAGCTGGAA TAGCGGCGTG CTTGGCGGAC
5160


 


GTGATCACCT TCCCGCTGGA CACGGCCAAA GTCCGGCTCC AGGTAGCTAG GCAGAGGGGT
5220


 


AAGACAAGGG GTCTCAGGAC AGAGGGGACG CTGTTGCGTG CATTCCATTT ATTCTCTGCT
5280


 


TTGGTGTAAC CACTGTTTCT AGGTAGGGTA GGTGACCTTC CAAAGCAGTC TGGCCTTGTC
5340


 


CCAGGGCTGG TGCTTTAGGA TGGGAAACTG GAACTTTTTC TGGGATTAGC TGAAGAACCA
5400


 


CCAGGGCCAC AGAGAATGGG TTGACCATGA CTACTACCAA ATTCTCCCAA AATTTAGGGT
5460


 


GCACTTAGTA TTTTAAGAGC TGAGAATATT GGCCTCTCCT GAGTTTACTA GTCAGGTGCT
5520


 


TTTTCCTTTC TTTGATTCTT CGGGGGTTCT GTCCTATCCT ACTGCCCTAG GGGTTCTGGA
5580


 


GAGTTCCTGG GGAGGGGGAT ATTCAAAATG TGCATTGTAG CCAGCCTCCC TCCATCTGCG
5640


 


CGTGAGCGAA CACACACACA CACACACACA CACACACACA CACACACACA CACACACGGT
5700


 


AGAGGGAGGT GGATGGAAGA GGAATGTTGC TGAGAAAAGA AACGGAAAAT AGGAACACAG
5760


 


GGGGAAATCT TGGCTTAAGA GTGAACTCAA TTTCGCTCCC TTCTGTTCTG CACCTTTCTT
5820


 


ATTTCCAGGT CCAAGGTGAA TGCCCGACGT CCAGTGTTAT TAGGTATAAA GGTGTCCTGG
5880


 


GAACAATCAC CGCTGTGGTA AAAACAGAAG GGCGGATGAA ACTCTACAGC GGGCTGCCTG
5940


 


CGGGGCTTCA GCGGCAAATC AGCTCCGCCT CTCTCAGGAT CGGCCTCTAC GACACGGTCC
6000


 


AGGAGTTCCT CACCGCAGGG AAAGAAAGTA AGCCGTGAGC GTTCCTGGGA GGGGCAGAAA
6060


 


AGCCTTGGGC TCCGCTCTGT TCCAAAAAGT GTAACACACA GAGGAGTGGT TTTCATAACA
6120


 


AATTGGCGAG AAAACATTCA TATTTGAACT CTCCCTTCCC CAAACATTAG CTCATTGTTC
6180


 


ATAGAAAAAA GTATGCAAAA TCGATTTTTT AGATGCAGAT ATATACTTGT AAAGGTCACC
6240


 


CAGTCATGGA AGTTTTGTGC CCAGTTTGGA TCTCCATCTG GAGAATATGG GTGGGCTACA
6300


 


GAAAAATGTT TAACTTAAAG TTCTCCAAAG AGGGAAGTAT ATCAGAAACA TCTATGGAGC
6360


 


TTGTCAGAAA TCCAAACGAG GACTACCATG GTCCTCTGAG TCTGAATCCT CAGGCTAGAG
6420


 


ACCAGAGTGT CTTTCCACAA GCTTCCCTCA TCATTTGTGT ATGCAACAAA GTTCAAAGCC
6480


 


TTCTGTTTGA AGCAAAGAAA GCCAGACTTT GTGAAGAGAG TTGAAAGGAC AGGAAAAGAC
6540


 


ATATTTCCTC TTAAGAGGTT CCTCATCAGG TCCAGGAAAG ACCAGAGCAG AAAAAGTGGA
6600


 


CGAATGCTGC AGGGAGTTTG TTTAGGGGAA AAAGAAAAGG AAACATATTT CCTGAGTGCC
6660


 


AGTGCACTCT AAGAATTCCT GTCACTTTAG GTAGCATTTA TTTGAGGGCT TAACTATGAA
6720


 


CCAGACATTG TTCTAAGTGC TTCAGATACA TTATAACTGG AAGGGTATTA GTACCATTAT
6780


 


CCCTTGGCAG ATGGGAAAAC TGAACACAGA GCAGATTCAT CACTTGCCCA AGGTCACACA
6840


 


GCTGGGAGGG GGCAGAGCCA GGGTTCAAAC CCAGGCAGTC TGGCCTCGGA CTCCAGGCTC
6900


 


CTAACCCTGT TCTCTACTGC CTTCTGCACT TCTCATATGA TTCTGCCCAT CATTCAAACC
6960


 


GCACAACACT GCTGTGAGTA AAAAGTGTTA GCCGAATATC AGGGTAGTTA AGTAACATGC
7020


 


ACAAAATCAC ACAGCTAATC AACATCAGAG GCACTTTCAT GTGGAGTAGA CAAGCCAGAG
7080


 


AGAAGATGTG CTGATGGCAC AATGAATACA TTAAGTGAAA TCCACCTTGT AGATTTCATC
7140


 


ATTTCTGCTG TGAGTAACCT TCAATACTAT AATTTTATGG GATAATTTAT AAATGTTGTC
7200


 


TATACAAATA TATAAGTTAT ACTTATCCAC ACAAGTACTT TCAAAGTGAA GATAAAGTCT
7260


 


GGATGTTACT AGATCAAAAC TGCATTTTTT TATTTATAGA TGTAGCAAGA GAGGAAACAC
7320


 


AAAGGAGGTA AAGCTGCCCG TTCAGGTGGT TTTCTTCACA GATTGACTGT TCTACCAATT
7380


 


GTTGTGGACT TTGGGCACCA AATTAATAGG ATATATGTTG GCAGTGTTCT ATGTTATATA
7440


 


GATTCAGTTT ATTTAGTAGG CTTTATTGAA CTGCCATGTG CCAGTAACTA TGTTAGATGT
7500


 


TTAGATGGCA GATGTGTCTC TAGACAGAGC TTACAGTTGA GAGTATGGGT TGTGTGGGGA
7560


 


GAAGTGAATA GATGACTATA TTCCATGATA CATGCTGTAT TACAATACAG TCCTACTTCA
7620


 


CTTAACGATG GGGATACATT CTCAGAAATG AGTTAGGAGG CAAATTGGTT GTTGAATGAA
7680


 


CATCACAGAG AGCACTTACA CAAACCTAGA TGGCATAGCC ACACCTAGGC TATATGGTAT
7740


 


AATCTATTGC TCCTAGGCTA CAAACCTGTG CAGCATGTTG GTATTGAATA CTACAGGCAA
7800


 


TTGTTACATA AAGTTAAGTG TTTGTGTACC TAAAAATAGA AAAGGTAATG CATTACACTA
7860


 


CAGTCTTATG GGGCTGGGAT GTCACTAGGT GATAGGAATT TTTCAGCTCT GTTCTAATCT
7920


 


TACGGGACCA CCATCATGTA TGCAGCACAT GACTAACTGT AATTACAAGA TGGTGGCTAT
7980


 


ATTAAACAGA ACTACTTAAG CTAGCCATGG AGGTATGGTC CGTGAGATTT TCCTGAAGAA
8040


 


TTAACGTCTG GATCAATTCT GGAAGGGCCA GCAGGAGTAC TCCAGGCAAA GGGGTGAGAA
8100


 


AGGAGCTTCC AAGTAGAGTG AAGGTCATGT GCAAAGACTC AGTGAGGAGT CGAGTGAACA
8160


 


TAGCACAGGG AGGACATGTT GGTGAGGAAG GAGGGGTGAA GCCACAGAGA CAGGAGGGAG
8220


 


CCAGATGACA GAAGGCCTTG CAGGCGGTGC TAAGGAGTTT GGATTTTATC CTTACAGTGG
8280


 


TGGGAAGTCA TTGTAAAAAT ATTAAGCAAG GGAGTGGCAT AAACAATTTA CATTTTCAAA
8340


 


AGATCACTTT GGCAGCAGAT AGAGTATATA TGTAAAAGGA GTAAGAAAGA GGTAAGTTAG
8400


 


AAAGCAAGAA ATGATCAGGG TATGCCCTAA AACACTGGCA ATAGGGAAAA AGAGATGTCA
8460


 


ATCAGAAAGA TTGAGAAAGT ATAATTGAAT TGACTTGGTG AACAAATAGA AGTAAGGCAT
8520


 


AAGGGACAGG TAGAAATATG AGATGACTTC CAAGTTTCTG TTTAAAGATA CCCTTTATTG
8580


 


AGAGAGGATG TATAGAAGCT GTCTTAGGGG GAAGACAAGA AATTTGGTTT AGGCCATGTC
8640


 


AACAGGTAAT GGCCAGTAGG CACATGATTC AGTTTATTTA GTGGGCTCCT TTTAGGAGAA
8700


 


AATCTGAGCC AGATTCCAGG AAGTCACAGC AGGGACTACC AATAGGGTCA AACAGCAGAG
8760


 


AGTGTGGAAA GGACTGAAAA GTGATCATTG TACATAACAA ATAGAAGCTC ACTGATTTTC
8820


 


TAGCAAAAAC ATCTTCAGCA GAGTAGCGTG GTATAAGCTA TATTGTAGGG GACTGAGGAA
8880


 


GAAATGGGCT CTGAGAAGTA AAGACAAACA ATATGTTTTG TAAATAAATT TCTTTTAGTT
8940


 


CTTAAAAAAA AAGCCTCTTT TCCAGCTTGA TTGGGAAGTG AAGAGAGGGA TTTGAAAGTT
9000


 


GGAGATTGGA GGATAGGATG AGTACATCAA GATACACTAC GTTGTAGTGC AGTGCATTAC
9060


 


AAATGTGAGC TAAAAGTGAA GGCATTTGTA ATCATATGAT ATTGCTAATT AAAAGACAGC
9120


 


TGTCAGTCAT ATGCCCAGCT CCTGGTAAAG CATGATGAGA AGAGTACAAT CATGGTAGTG
9180


 


ATTTAAAAAT TGCTGCCAGT TTTGTGGATT TTCTTTATGC TAGACAGTGT AAGCTCTTTA
9240


 


TCAATATTAT TTAACTCACA CAACTCTAAG AGGTAGATAT TATTATCCCT TTTTGACAAA
9300


 


TTAGGAAACA GAATTATAAT GACTGAGAAA GTCTCTGCTG AGTAAATGTT ACTGAACCTT
9360


 


AATTTTATGT TTACTTAATG ATAGAAATGA ATATTGGGCT TCAAGACTAT TTGTACTTAA
9420


 


TGAAATCTGT CTTGAGCAAC ATAAGCTATT TTTTTCAAAA TTTTAAGACA AAAATCACTT
9480


 


TCTTCTCTCC TGTCTTCTTA TTTTTGTTCC CTTCACATGT TGTAGCCTAA CACTACTTGA
9540


 


TGGCCCATTT TGGTGCAGTT TGTCCACTGG GCTTCATCTA AGGCCACCAA GTCCCATAAT
9600


 


TAACATGATC ATTCGTGGGA GAAAGATCAA GCCTCATTGG TGATGGGTGC CTCCTCACAG
9660


 


TCGGATAATA CTGAAAAGAG AGCTAAATGT GGGAAAGAAC CAAGTTGAAC ACAGGAAAGA
9720


 


ATCAGGCCAC TGTGAAAATA AGCATTGTGT TTTCTTGTTC CTTGAAAGTC TTCATTTTTA
9780


 


AAAAATTTCA GACACCTGAA GTTTTCTAGC CTTACTCTGA GTTGACGCAC ATTTAGTACA
9840


 


TGATCAACAC ATAAACAAGC ATTAGAGAAA TAGAAAAGCT GTAAGAATAC AAAAATATGG
9900


 


GCCAGGTGGG TGGCTCATAC CTGTAATCCT AGCACTTTGG GAGGCCGAGG CAGACGGATC
9960


 


ACCTGAGGTC AGGAGTTCAA GACTAGCCTG GCCAATATAG TGAAACCCTG TCTCTACTAA
10020


 


AAATACAAAA CTTAGCAGGC TGTGGTGGCA CGTGCCTATA ATCCCAGCTA CTTGGGAGGC
10080


 


TGAGGCAGGA GAATCTCTTG AACCCGGGAG GCGGAGATTG CAGTGAGCCA AGATCACACC
10140


 


ACTGCACTCT AGCCTAGATA ACAGAGCAAG ACTCCATCTC AAAAAAAAAA AAAATACAAA
10200


 


AATATGAACC ACTGAAAATT AAAAAGACAT GCATGCATTC TAGGTCTTTA ATTTTTTTTC
10260


 


TTAATAATTT TTTTTCTCTC TGGATAGCAG CACCTAGTTT AGGAAGCAAG ATTTTAGCTG
10320


 


GTCTAACGAC TGGAGGAGTG GCAGTATTCA TTGGGCAACC CACAGAGGTC GTGAAAGTCA
10380


 


GACTTCAAGC ACAGAGCCAT CTCCACGGAA TCAAACCTCG CTACACGGGG ACTTATAATG
10440


 


CGTACAGAAT AATAGCAACA ACCGAAGGCT TGACGGGTCT TTGGAAAGGT AACTAACTTC
10500


 


AAAATGGGTT TTATAACCAC CAAAGCACAT ACATACAACT AGCAACTTAT TGTAAAGTAG
10560


 


AGTTAATAAA CATTTTCTTT TTTTTTTTCC CCAGGGACTA CTCCCAATCT GATGAGAAGT
10620


 


GTCATCATCA ATTGTACAGA GCTAGTAACA TATGATCTAA TGAAGGAGGC CTTTGTGAAA
10680


 


AACAACATAT TAGCAGGTAA CTTCCCATTT CATATAACAA ACAGGTCTGC ACCTTTAGAA
10740


 


GTTCATCTTG GAGCTTCTGC AGCCACCTTA TACTCAATCT CTTAACTCCA ATAGTTTTCT
10800


 


CTTTTTAAAA ATTAAGTAAT TTTGAACCAT ATATAACTTT GTGAGAAGCA GGAAAAGACC
10860


 


AAAATATTAA GTTTAAGAAG TTTTGCCACA ACAAAAATAT TTTGCAACAA AAATAACAGG
10920


 


CAATTTCATG TCAGCATTAT TCTCATTTAA TACTAATATA TGGGACTTTT GTTAGAATCT
10980


 


TATTCTTTAT ACAGCAGAAT TCAGGAGGTA AGTCCATCCT GCATACTATA TCCAAAAGAT
11040


 


CTAGTTATAA AAGGAGCTTA TCAGTGGTCT CATCCAAAAA GTAATACCAT AAGATAGGTT
11100


 


CTTAAAAATA ATATTCTAAC AACTTCTAGA GACATTGAAA TTTCCCTTAT TTCAATAAAA
11160


 


AAGTATTAGA TGCTCATATA TTAGGCATTA TTACAGGCCT TAAAGGCACA GAGGAAACTA
11220


 


ACAGTTTACT TTCCTAAAGT GTTAACAATC TATTAAGCCA TTTACTCTTT ACCTTCTTTT
11280


 


TCTAGTGCAA TACCTTTCTT ATTTTATTTT ATTTATTTAT AAGACATCTT CATTGACCTA
11340


 


CTGTTATCAA TAGGTTTATA AAGATATGAC AGATAACTAA ATTGCAAGCC CCCAAAAGTC
11400


 


TGATGTTGAC CTGTTTCATC GATCCATTTT AGATGACGTC CCCTGCCACT TGGTGTCGGC
11460


 


TCTTATCGCT GGATTTTGCG CAACAGCTAT GTCCTCCCCG GTGGATGTAG TAAAAACCAG
11520


 


ATTTATTAAT TCTCCACCAG GACAGTACAA AAGTGTGCCC AACTGTGCAA TGAAAGTGTT
11580


 


CACTAACGAA GGACCAACGG CTTTCTTCAA GGGGTAAGAT ATGATCTTGT GTATCTGTAA
11640


 


TGTGTTCTGG CTGTCTGTGT GCTTTGGGAC ACTCTCATGT CAAGCAACCG ACATTTAGCT
11700


 


TACAAGCCTT AGTATATTCA TATACTTAGT ATTGACTTTT CCTTGCCACA GATTTCTCCA
11760


 


ATCCACCAAT TCCACTGTGC CAGAAAGTAA AAAGCCATGA TATTCAAATT TTCTCAACTT
11820


 


TGATCAAAGG CTCATTCAAG ACCAGTGCCT TTTCCACTGG TCCCAATCTA CTGGAAATGC
11880


 


AGACAGTATT TTGCCTTCTC TGGGCAAGAA AGTTATAAAG TAGAGGGAAA TCATAATAGA
11940


 


GAGCTATGAG AGAACAAGAT TTGATTTGAT TTAATTTGAT GGACTCAAGT TTTAACATTG
12000


 


TAAAACTAGA GATAAGACAT CACCACCAAT CTAGAAAAGT GATGCAGAAA AGTATTTGAT
12060


 


TTGGGTAATT ATTACACTCA CCTAGAAACA AGTGTTGTGT AATAGATTAC ATATTTCCAT
12120


 


AATGCAATGT TGTATCAGAA ACTACCTTCC TAAGAAAATA TAGTATGGGC TCGGCGTGGT
12180


 


GGCTCGCACC TGTAATCCCA GCACTTTGGG AGATGGAGGC AGGAGGATCA CTTGAGCCCA
12240


 


GACTGGGCAA CAAAGCGAGA CCCTGTCTCA ACAAAAAATT TAAAAATTAG CTGAGTGTGG
12300


 


TGGCACGCAC TGATGGTCCC CTCTACTTGG GAAGCTGAGG CAAGAGGATC TCCTGAGCCC
12360


 


AGGAGTTCAA GGTTTCAGCG AGCTATGATT GTGCCACTGC ACTCCAGCCT GGGAGACAGA
12420


 


GCAAGTCCCT GTCTCAAAAA AGAAGAAGGA GAAGGAGGAG AAAATACAGT ATTAAGTAAT
12480


 


CTGTCAATAT ATTCCACAAG GATTACACTA GTGGTTTAAT AATAAAATTA TATTACCTTT
12540


 


TTAAATTGTA AGGCCATTCC TCAAGCTTTA TAAATTAAGC ATGAATGCAT CATACACATT
12600


 


TTATAAAAAG TTCCAACTCA TCATAATCTG TACTTATGAT ACATTAATAC AAATGAAGTT
12660


 


CATTATAAAA TTAACTTAAA ATGGATATAC CAGTTATTAA ACCATTAACC ATTTAATAAT
12720


 


TTTATTTTTT TCAAATTTAA AAACCTTTTG GGGAAGAAAT ACTACAACAT GGATGAACCT
12780


 


TGAAAACGTT ATGCTAAGTG AAATAAGCCA GACACAAAAG GACAAATACT GTATGATTAC
12840


 


ACTTAAATGA GGTACCTAGA GTAGTCAAAT TCATAGAGAC AGAAAGAATA GAAGTTACCA
12900


 


GGGGCTGGAG GTAGGAAAAA ATGGAGAGCT GTTTAATGGG TAGAGAGTTT CTTTTTGGGG
12960


 


TGACAAAAAG GTTCTAGAGA TGGATAGTGG TGATGGTTAC ACACAATGTG TGTGTACTTA
13020


 


ATGCTACTGA AATGTAATTT TATGATTTTT TTTTTTTGCA GCAAAATACC CCACATTGGG
13080


 


AAGTGAAGAG AAACATGTTA AGAGACTTGA AGGAAAAAAA TTGGGGCAGA GGGGTGTTTT
13140


 


TTATAGGTTA AACAATAAAA GCCATTTAAA CAGTAACAAT TTCTCTAAGG ACAAGAATCG
13200


 


TCAAGATTGA GACAGCACTG ATTTCTTGAC TCTACTCAAT ACTTCTTTGG TTTCTCTTCT
13260


 


TCCTTCCCCC TTCTAATAGT TTCCTACCTC CCATTCAGAA AGCAAAGCAA AACAAGCAAA
13320


 


AATTCCCCCT TCCCTCAAAA AAGGAAAGAG TTTTTGAAAA AGTTCATGTC AGTGAAGAAA
13380


 


AGACATGTTT TGGGAGTGAA GGATATTTGT GGATTTGTAT AGATGTGATC ATCAGGGCTG
13440


 


TGTTGTTTTG AAGTAATATA GGACATCTAG AGGAAAATTT ATTTTCAGCA GAGGAGGGAA
13500


 


AGATGAAGAG TAGGTACTTT TAAGCATCTT CACTTGAGGA GTGGCAAAAT GAGAAGCATA
13560


 


ACCTGCTATA ATCACTTTAA GAATTTCAGG CTGAGTGTGG TGGTGCAGTC TCTAGTCCCA
13620


 


GTTACTCCAG GAGGCTCAGG TGGGAGGATC ACTTAAGCCC AGGAGCTCGA GGTTGCAGTG
13680


 


AGCTATGATT ACACTACTGC ATTCCAGCCT GGGCGGCAGG GTGAAGCCTC ATCTCAAAAA
13740


 


TTAAAAAAAA AAAAAATCAA ACAAATTAAT CGAACGATGA CATGCACTTT TCTAGGTTGG
13800


 


TACCTTCCTT CTTGCGACTT GGATCCTGGA ACGTCATTAT GTTTGTGTGC TTTGAACAAC
13860


 


TGAAACGAGA ACTGTCAAAG TCAAGGCAGA CTATGGACTG TGCCACATAA TCAGCTTCAA
13920


 


GAAAATGATG TAACATACCA GTGGGAATCT TGCTGACTGG ATCATAAAAA CAAACAAAAC
13980


 


TTATTCACTT ATTTTAACCT AAAAAGATAA AGGAATTTTG GCAGAGAATT TTGGACTTTT
14040


 


TTATATAAAA AAGAGGAAAA TTAATGCCTA TTTCATATAA CTTTTTTTTT TTCTCAGTGT
14100


 


CTTAAGAAGG GGAAAGCAAA ACATTCAGCA TATACCCTGG CAAATGTAAT GCAGATAAGC
14160


 


TACTGCATTT GACCATTTCT GGAGTGCAAT TGTGTGAATG AATGTGAAGA ACTTTAACAT
14220


 


GTTTTAATTA CAATTCCAAC TGGTGGAAAA GAAACTGAGT GAAATGCAGT TTATATTTAT
14280


 


AAATACTTAA AAATGAAGTT ATTAAAAATA TTAGTTTTTA TTAACCACAG TTGTCAGTTA
14340


 


ATATATTCAA TAAAGTATTG CTAATACCTT TTAAAGTTTG TCTTTTGAGA TCTATACCTG
14400


 


GGTGTAAGAG TCAAGTTCAC TAGAATACAA GACTGCCCAA TAGCAAATGC AGGTCTTTAG
14460


 


AATCATAGGC ATGAACCTAC TCTGAATGTT ATTAGTATAG ATTTTTAATG TTTAGAGTCC
14520


 


AGATTTGATG ACATCTCTAA CAACTTCTAA TCTAAGACAC TATATTCATT TTGGCAGGAT
14580


 


TGCTACTAGA GTCTTGGTAT CTGTGCTAGC ATCACATAAT TTTAGAGCTG GAGGGTACTT
14640


 


CTGGGAAGAC AGAGGAACAG TTTGAGATTC CTACTGAGAT GAAAACGAAT CTTCATGGAA
14700


 


TCTTTCAGCA AAGCCAAATT CAAATTCATC ATTAGCACCT GTAGTAACCT TTTCAATGCC
14760


 


TACAAACTGC ATGCAGAAGA GATAGGGAAA CAGTAAAACA GATATTAAAA GAAGTTTTTA
14820


 


AGACAAAGCC CAGCCTGATT TTAAGCTAAA TCCAAGGATT GGCAGCTTGG ATGAGCAGGA
14880


 


AGGTTACAGG CTGCCAGACA TCATTCTAGT TCTGTTTTAA TCAACTCCAT GTTACATTTA
14940


 


CTATCAGGGA TTCTCACCTC ACCCTCATGC ATGTCTTCCC CATTCATTAC CCGCAAAAGT
15000


 


GTCTTGTAGC AGATGTCTTC TGTGTCCCAT ACATACCATT TTGCTCTTTA GTGCTTGCTG
15060


 


GCCTGACTTC CTATTGTCAT GTCAGCATCT GCCCTTTTTA GGGTCTCTGG CCACCAGAGC
15120


 


CAGCTTTACT CACCTGTGCA TGGCATTCTA GAAGAGCAGC AGGGAAAATA ACACAGCCCC
15180


 


AGTGCAGCCC TTAACCACCA ATAACTGGTA GTAGTTGGTG TACAAATATC TCAGTTCCCT
15240


 


CAACTGTCAG GTGGAATACC GCTGAGGGAT CAAACTCTAG TAACACACAG TAGTGTTTTG
15300


 


CTTACTATGG TTAACTAAAA AATCACAGGG TCTTCATGCA TTTGGAAAGG ATACTTTATT
15360


 


TCTTACAAAG GGTTACAGCC TACAAGGTGG TCATTCTGCA GGCTAGAAAG CGTAACCTCC
15420


 


AGCAAAGACC GGAGGCAGGC ACTTCTAGGG AAGGAAGAGT AAGACAGAAA TTTAAATTGA
15480


 


ATGGGTTGGC CAAGTATACA TATTCAACAG GCTACAGGTG GATTCATGAA TATTCATGAA
15540


 


GGCAGTCCTG ATGCATGCAT GTTACACCTT GGGGTGGAGG CTTAACATTT AAATGTATTA
15600


 


CAGTTAGGCC CTATACATGA AAAGGTGAAG CAGTAACACG AAGGCACACA ATGCACCATT
15660


 


TCTGTAAACA GGCCAGAGCC AGTTCACAGT GGTTGGTCTC TTATCATGAG AAAGCTACTA
15720


 


AAATCCTCTT GTCCAGTTAA AACTGTAGTT ATGGCTGGTG GAAAATGGGC TGGAGTCAGT
15780


 


CAACACTTGG TGAAGCTGCA GTTGCTTCAG ACACTCAAGG CCAGTGTTTG TTTAGCTGCT
15840


 


CGAGAAAAAG AAAAATCTTG TGGCAGTTAG AACATAGTTT ATTCTTTAAG TGTAGGAGTG
15900


 


TGTGACTTAA //







The nucleic acid sequence of the 2,113 base pair (bp) transcript ENST00000310473 of the human UCP2 gene is as follows (Eight Coding Exons in capital letters) (SEQ ID NO: 366-382):


























SEQ



Exon/


Start
End


ID


No
Intron
Start
End
Phase
Phase
Length
Sequence
NO








5′





...aatcgacagc gaggccggtc gcgaggcccc
366



upstream





agtcccgccc tgcaggagcc




sequence









 


1
ENSE00002
73,694,352
73,693,766
—
—
587
AGCCGCGCGC TCGCTCGCAG GAGGGTGGGT
367



287650





AGTTTGCCCAGCGTAGGGGG










GCTGGGCCCA TAAAAGAGGA AGTGCACTTA AGACACGGCC










CCGCTGGACG CTGTTAGAAA CCGTCCTGGC TGGGAAGGCA










AGAGGTGTGT GACTGGACAA GACTTGTTTC TGGCGGTCAG










TCTTGCCATC










CTCACAGAGG TTGGCGGCCC GAGAGAGTGT GAGGCAGAGG










CGGGGAGTGG CAAGGGAGTG ACCATCTCGG GGAACGAAGG










AGTAAACGCG GTGATGGGAC GCACGGAAAC GGGAGTGGAG










AAAGTCATGG AGAGAACCCT AGGCGGGGCG GTCCCCGCGG










AAAGGCGGCT GCTCCAGGGT CTCCGCACCC AAGTAGGAGC










TGGCAGGCCC GGCCCCGCCC CGCAGGCCCC ACCCCGGGCC










CCGCCCCCGA GGCTTAAGCC GCGCCGCCGC CTGCGCGGAG










CCCCACTGCG










AAGCCCAGCT GCGCGCGCCT TGGGATTGAC TGTCCACGCT










CGCCCGGCTC










GTCCGACGCG CCCTCCGCCA GCCGACAGAC ACAGCCGCAC










GCACTGCCGT GTTCTCCCTG CGGCTCG



 



Intron
73,693,765
73,692,678


1,088
gtgagcctgg ccccagccct gcgcc...actct
368



1-2





ctgcctttgc tcacccacag



 


2
ENSE00001
73,692,677
73,692,521
—
—
157
GACACATAGT ATGACCATTA GGTGTTTCGT CTCCCACCCA
369



184362





TTTTCTATGG










AAAACCAAGG GGATCGGGCC ATGATAGCCA CTGGCAGCTT










TGAAGAACGG GACACCTTTA GAGAAGCTTG ATCTTGGAGG










CCTCACCGTG AGACCTTACA AAGCCGG



 



Intron
73,692,520
73,689,523


2,998
gtaagagtcc agtccaagga agagg...tgggg
370



2-3





cttttctcct cttggcttag



 


3
ENSE00001
73,689,522
73,689,298
—
0
225
ATTCCGGCAG AGTTCCTCTA TCTCGTCTTG TTGCTGATTA
371



184370





AAGGTGCCCC










TGTCTCCAGT TTTTCTCCAT CTCCTGGGAC GTAGCAGGAA










ATCAGCATCA










TGGTTGGGTT CAAGGCCACA GATGTGCCCC CTACTGCCAC










TGTGAAGTTT










CTTGGGGCTG GCACAGCTGC CTGCATCGCA GATCTCATCA










CCTTTCCTCT










GGATACTGCT AAAGTCCGGT TACAG



 



Intron
73,689,298
73,689,142


156
gtgaggggat gaagcctggg agtct...tagct
372



3-4





accctgtctt ggccttgcag



 


4
ENSE00001
73,689,141
73,688,931
0
1
211
ATCCAAGGAG AAAGTCAGGG GCCAGTGCGC GCTACAGCCA
373



252503





GCGCCCAGTA CCGCGGTGTG ATGGGCACCA TTCTGACCAT










GGTGCGTACT GAGGGCCCCC GAAGCCTCTA CAATGGGCTG










GTTGCCGGCC










TGCAGCGCCA AATGAGCTTT GCCTCTGTCC GCATCGGCCT










GTATGATTCT










GTCAAACAGT TCTACACCAA GGGCTCTGAG C



 
Intron
73,688,930
73,688,063


868
gtgagtatgg agcaagggtg taggc...cactg
374



4-5





accccatggc tcgcccacag



 


5
ENSE00001
73,688,062
73,687,868
1
1
195
ATGCCAGCAT TGGGAGCCGC CTCCTAGCAG GCAGCACCAC
375



184355





AGGTGCCCTG GCTGTGGCTG TGGCCCAGCC CACGGATGTG










GTAAAGGTCC GATTCCAAGC TCAGGCCCGG GCTGGAGGTG










GTCGGAGATA CCAAAGCACC GTCAATGCCT ACAAGACCAT










TGCCCGAGAG GAAGGGTTCC GGGGCCTCTG GAAAG



 


 
Intron
73,687,867
73,687,788


80
gtgtgtacca gttgttttcc cttcc...accca
376



5-6





ggatcttcct cctcctacag



 


6
ENSE00003
73,687,787
73,687,686
1
1
102
GGACCTCTCC CAATGTTGCT CGTAATGCCA TTGTCAACTG
377









TGCTGAGCTG




147097





GTGACCTATG ACCTCATCAA GGATGCCCTC CTGAAAGCCA










ACCTCATGAC










AG



 


 
Intron
73,687,685
73,686,717


969
gtgagtcatg aggtagacgg tgctg...tgcct
378



6-7





tgcctgctcc tccttggcag



 


7
ENSE00001
73,686,716
73,686,536
1
2
181
ATGACCTCCC TTGCCACTTC ACTTCTGCCT TTGGGGCAGG
379



184349





CTTCTGCACC










ACTGTCATCG CCTCCCCTGT AGACGTGGTC AAGACGAGAT










ACATGAACTC










TGCCCTGGGC CAGTACAGTA GCGCTGGCCA CTGTGCCCTT










ACCATGCTCC










AGAAGGAGGG GCCCCGAGCC TTCTACAAAGG



 


 
Intron
73,685,535
73,686,167


369
gtgagcctct ggtcctcccc accca...atgac
380



7-8





ctgtgatttt tctcctctag



 


8
ENSE00001
73,685,166
73,685,712
2
—
455
GTTCATGCCC TCCTTTCTCC GCTTGGGTTC CTGGAACGTG
381



184368





GTGATGTTCG










TCACCTATGA GCAGCTGAAA CGAGCCCTCA TGGCTGCCTG










CACTTCCCGA










GAGGCTCCCT TCTGAGCCTC TCCTGCTGCT GACCTGATCA










CCTCTGGCTT










TGTCTCTAGC CGGGCCATGC TTTCCTTTTC TTCCTTCTTT










CTCTTCCCTC










CTTCCCTTCT CTCCTTCCCT CTTTCCCCAC CTCTTCCTTC










CGCTCCTTTA CCTACCACCT TCCCTCTTTC TACATTCTCA










TCTACTCATT










GTCTCAGTGC TGGTGGAGTT GACATTTGAC AGTGTGGGAG










GCCTCGTACC










AGCCAGGATC CCAAGCGTCC CGTCCCTTGG AAAGTTCAGC










CAGAATCTTC GTCCTGCCCC CGACAGCCCA GCCTAGCCCA










CTTGTCATCC










ATAAAGCAAG CTCAACCTTG GCGTC



 



3′ down-





tcctccctct cttgtagctc ttaccagagg tcttggtcca
382



stream





atggcctttt...




sequence









The nucleic acid sequence of the 15,174 base pair (bp) of the human UCP2 gene (ENSG00000175567), including 5,000 bp 5′UTR and 2,000 bp 3′UTR, is as follows (Exons are highlighted) [SEQ ID NO: 383]:








TCCAGCCTGG GCAACAAGAG TGAAACTCGG TCTCAAAAAA AAAAAAAAGA GAAGAAGAAG      60



 


AAAGAAAACT AGGTGGAGTG TGGTGGCTTG CACCTATAAT CCCAGCACTT TGGGAGGCCG     120


 


AGGTGGGTGG ATCTATTGAG GCTAGGAGTT CAAGATCAAC CTGCCAACAT GACGAAACCC     180


 


CACCTCTACT AAAAATACAA AAAATTAGCA CGGCGTGGTG TGTGTGCCTG TAATCCTAGC     240


 


TACTTGGAAG GCTGAGGCAG GAATCGCTTG AACCTGGGGG GCAGAGGTTG CAGTGAGCCA     300


 


AGATCTTGCC ACTGCACTCC AGGCTGGGCG ACACAGCACA ACTCTATCTC AAAAATACAA     360


 


AGAAAAAACA AAAGAAAACT AATATATCAA AATAATTTCT AGTTAGTTGG ATTCACTCAC     420


 


TTATTCATTC AATGACTTAT TGAATTATCA TATATTACTA GTGCTTTTTA ATACATACCT     480


 


TCTACAATTT TTCAACTGAA AATTACTTCA TTGATCAGGG CTCTTTAAAC TGATCTCCAT     540


 


TTGCATTGTT TTACTAACTA TAGTTATTAT TCATGTATTA GCACTCTGAG CCTACTGTAA     600


 


TGATGTGTAC CTTAATAAAG AACTGAATAT TTGTAATGGC TGGCAGTGAA TTTAGTAGTT     660


 


CTTGAATTTA GAGCTCAAAA TATGGGAGTA ATTTGCTGCT TTATTTCCTT TGAGAGGTAA     720


 


TAGAGGAAAA ACAGAATCTA ATAACAATCA CAGATTTTCG GGAAAGCACT GTAAAACCAT     780


 


ATGATCAATT CTAGCTTCTT ATGTAAACAT GGAAAGATTG CCAGCTGAAC ACCTGTCATG     840


 


CTCTAAGAAG TTGGGGAGAA TTTGCATTTT TAGAACTGTG AGCAAAATGA GAACGACTGC     900


 


TATGTTCATG CTTTGTGAAT TTAGCTTTAT TTCATTCACA CAATTCATGG GAAAAAATGC     960


 


ATCTTTTAAC TCGGTGTTTT TCAATTCAAC TTTTAAAATA CAGGAGTGGG CCAGACCCGG    1020


 


TGGCTCACAC CTGTAATCTC ATCACTTTGG GAGGCCGAGG CAGGTGGACC ACAAGGTCAA    1080


 


GAGATAGACA CCATCCTGGC CAACATGGTG AAACCCCATC TCCACTAAAA ATACAAAAAT    1140


 


TAGCTGGGCA TGTTGGCACG TACCTGTAAT CCCAGCTACT CGGGAGACTG AGGCAGGAGA    1200


 


ATCGCTTGAA CCTGGGAGAT GGAGGTTACA GTAAGCCGAG ATCGCGCCAC TGCACTCCAG    1260


 


CCTGGCGACA GAGCAAGACT CCATCTCAAA AAAAATACAA AAAAATACAA AAAAATACAA    1320


 


AAAAAACCAG GATGTGTTAC CAAGGAAAAT TCATTTACAA TGGTTAATTA TGTGACAAAC    1380


 


ATGTCAAGTA ATTCCATCTG GCTTTGTGTC ACCATTTCCC CACCCTTTTT TCAGAAACCA    1440


 


AAACCAAGAA GAAGAACAAA CATCAAAATG GACATGGAAA TTAACAAATA TATGATTCAA    1500


 


TTTAATCTCC TAAGAGGTTT TTTAAAATTA TTTTATTTTG AGACGGAGTC TTGCTCTGTC    1560


 


GCCAGGCTGG AGTGCAGTGG CAGGATCTCA GCTCACTGCA ACCTCCATCT CCCAGGTTCA    1620


 


AGCGATTCTC CTGCTTCAGC CTCCCAAGTA GCTGGAACTA CAGGCAAGCA CCACCACACC    1680


 


CAGCTAATGT TTGTATTTTT GGTAGAGATG GGGTTTCACC ATGTTGGCCA GGATGGTCTC    1740


 


GATCTCTTGA CCTCATGATC CACCCGCCTT GGCCTCCCAA AGTGCTGGGA TTACAGGTAT    1800


 


TTTTTATTTT TTTTGAGACA GGGTCACCCT GTCACCCAGG CTGGAGTGTA GTGGCACAAT    1860


 


CATGGCTCAC TGCAGCCTCA ACCTCCCAGG CTCAGGTGAT CCTCCATGTC AGCCTCCCAA    1920


 


GTAGCTGGAA CTATAGGCGT GCAACACCAT GCCCAGCTAA TTTTTGTATT TTTTGTAGAG    1980


 


ACAGGGATTT GCCATGTTGG CCAGGCTGGT CTTCAACTCC TGGCCTCAAG TGATCCACCC    2040


 


GTCTCAACCT CCCAAACTGC TAGGATTACA GGTGTGAGCC ACCGTGCCCC ATCTCATCTG    2100


 


CTAAGTGGGT TTAAAGAAAT TCAGTTTCAT GTCAATTTTT AAAATGTATG GTTATCAAAT    2160


 


TCGACTTCTT TTTAAAAATG CAATCAGATA ACTGTATGCT TGTTTGATGA GGGGAGGAAA    2220


 


GTTAATATAG CCAATCTACT CAATATTTTT AGCAGAAATT ATCAGAGACT AAGGAAATGT    2280


 


TTAAGTTTTT CTCATGTTGG TTTTAATTAC CTAATGTTTT CAGTTTTCTC TTTCATTCTT    2340


 


GTGTCTTTTT TTCATTTTCA GTGTTTCAAA TACAGTTTGT ATTTAAAGAT TTAGAAGTTC    2400


 


CAAAACTGTA AGCACAGTGG ATTGTTTCCT GGGATGATGT TAAAATTATA CAACAAAATA    2460


 


TATGAAACTT TGTCAATTTG GTTATTGGCA CATACAAAAT ATTTACAAAT AAACGTGTGT    2520


 


GTGTGTGCGT GTACACACAA TTCAATGAAA TAGATGTGAA ACAAGTTTTC TTTTTTTTTT    2580


 


TTTTGAGACA GAGTCTTGCT CTGTCGCCCA GGCTGGAGTG CAATGTCGCA GTCTCAGCTC    2640


 


ACTGCAACCT CTGCCTCCCG GGTTCAAGCG ATTCTCCTGC CTCAGCCTCC CGAGTAGCTG    2700


 


GGACTACAGG CACCTACCAC CACTCCCAGC TAATTTTTGT GTTTTTAGTA GAGACAGGGT    2760


 


TTCACCATGT TAGCCAGGCT AGTCTCCAAC TCCTGACCTC AGGTGATCTG CCCGCCTCAG    2820


 


CCTCCCAAAG TGCTGGGATT GCAGGCGTGA GCCACCTCAC CTGGCTACAA GTTTTCAAAA    2880


 


TACATTTATC TGTACCCATA CATTCTCCAG TTTGTCCACA GGACATCTTA TGACTTGAGC    2940


 


AAGCTGCTAA AAATCCAAGG GTGCAGCGTT TGTATGTCTA TAGGATTGCT CAGATCTGCC    3000


 


CCCACCCTGA AAGAATTTAA GAGAATTTCT TGAGGCCAGG CACAGTGGCT CACACCTGTA    3060


 


ATTCCAGTAC TGTGAGAGTC CGAGGTCAGA GGACTGCTTG AGGCCAGGAG TTCAAGAGCA    3120


 


GCCTGGACAA CATAGGGAGA CCTGTCACTA CAAAGAATAA ATAAATTAGC CAGGCTTAGT    3180


 


GGCTCATCCC TGTGGTCCCA GCTACTAGGG AGGCAGAAGT AGGACTGCTT GTCCCAGGAG    3240


 


GTCAAGACTG CAGTGAGCTG AGACCCAGCC ACCTGCATTC CAGCCTGGGC AACAAAAAGA    3300


 


GACCCTGTCT CAAAAAATAA GTTAAATAAA TAAATAATAA AAATAGTTTA AACCCTAAAC    3360


 


ACATCTTCTT TTTCAAAGAG GACTTCTTAA GGACTTCATG CTGCGTCCTG TTGATCTCCA    3420


 


CTTCCCTTTT TCAGCGTCCA CACTTTTAAC AGTCTCTTTT GCCAAGGATA ATAAGTATAT    3480


 


AGTTTCTGGA ATCCAGATTC TTCCCTGTTT GGACAGCCAG GGGGACAATT TTTGGTCTGC    3540


 


AGGCCTTTGC ATCTGTTCTG CTGTTGCTCA GCAATCTCAC AGCAAATTTG CCGAGCCTCT    3600


 


CCGGAATGCA CAGCCAGACA GAGCTCAGCG CAAAAGCTAG AGAACCTGGC GGAGGGAGAC    3660


 


TCACAGTGCC ACAAAAAAAC TTTATCTTTT CTTTTTTTTT TTCTTTTCTT TCTTTCTCTT    3720


 


TCTTTCTTGT CTTTCTGTCT TTCCTCTCTC TCTCTCTGTC TTTCTTTCCT CTCTTTCTTT    3780


 


CTTTTTTCCT ACATGGCAAG ATCTCCTCAT GGCAGAAATA ATCTGCCTTG ACTTCTGTTT    3840


 


CCACGCTGCT TCTGCCAGGA CCATGCGCTC GGCGTGTTTT TCTTTCCGCT ATAATTATCC    3900


 


AGGCCCATCC CAGCTCTGGT CCCCTCAGCT GTTCCCTGGC AGTCCCTTCT GCTGGTGAAA    3960


 


ACACATATGG CGCCGGCCTG ACCAGGGTGT AAGTGTGTGA ATATCAGGAA GATGACTGAA    4020


 


CGTCTTTGGG ACTCCGTTTC CTCATTGTAA AATGGAGGTT AATACCAGCC TTCTTCTACT    4080


 


CCCCAAACGC ACGTGTTTGT CCCGGCCAGA GGGCCCAATT GTTGGCTGTT CACGCGTCAG    4140


 


TTACCCCCAC AGGACGGGTC AGCCAATTAA AGGCGAACCA GGCCCGGTCC ATCTCCTGAC    4200


 


GCCTTTTCTC ATCCCAGGGC TGGACAGGCA GCTGGCCTGG GCCCGGCTCT GCCTTGTCAC    4260


 


GTGCGGGGGC CGGCCCGTTT GCTTGTCTGT GTGTAGGAGC GTGAGGTCAC GCTGGGTGCT    4320


 


CCCGCCCCGC CGGGGCCTTT AGTGTCCTGG TCCCTAAACG CCAGGCCGCT CCACCGGGGG    4380


 


AGAAGGCGCG AACCCCAGCC GAGCCCAACG GCTGTTGTCG GTTGCCGGGC CACCTGTTGC    4440


 


TGCAGTTCTG ATTGGTTCCT TCCCCCGACA ACGCGGCGGC TGTAACCAAT CGACAGCGAG    4500


 


GCCGGTCGCG AGGCCCCAGT CCCGCCCTGC AGGAGCCAGC CGCGCGCTCG CTCGCAGGAG    4560


 


GGTGGGTAGT TTGCCCAGCG TAGGGGGGCT GGGCCCATAA AAGAGGAAGT GCACTTAAGA    4620


 


CACGGCCCCG CTGGACGCTG TTAGAAACCG TCCTGGCTGG GAAGGCAAGA GGTGTGTGAC    4680


 


TGGACAAGAC TTGTTTCTGG CGGTCAGTCT TGCCATCCTC ACAGAGGTTG GCGGCCCGAG    4740


 


AGAGTGTGAG GCAGAGGCGG GGAGTGGCAA GGGAGTGACC ATCTCGGGGA ACGAAGGAGT    4800


 


AAACGCGGTG ATGGGACGCA CGGAAACGGG AGTGGAGAAA GTCATGGAGA GAACCCTAGG    4860


 


CGGGGCGGTC CCCGCGGAAA GGCGGCTGCT CCAGGGTCTC CGCACCCAAG TAGGAGCTGG    4920


 


CAGGCCCGGC CCCGCCCCGC AGGCCCCACC CCGGGCCCCG CCCCCGAGGC TTAAGCCGCG    4980


 














CTGCCGTGTT CTCCCTGCGG CTCGGTGAGC CTGGCCCCAG CCCTGCGCCC TTTGCGCCCC    5160


 


CCACGCTTGT TCTGCGTGCG CTGCCCGCTC TTCCATTTAC CTTCTCTCCC ACCCAAGTTT    5220


 


GTACTCTTTT CTTTCTCTCG GTTTTATTTT TTGTTTTTGT TTGTTTGTTT GAGACAGGCT    5280


 


TTCGCTCTGT CTCCCAGGCT GGAGTGCAGT GGCGCGATCT CGGCTCACTG CAGCCTCCAC    5340


 


CTCCCAGGTT CAAGCGATCC GCCTGCCGAG TAGCTGGGAT TACAGGCGCC CGCCACCACG    5400


 


CCTGGCTAAT TTTTGTGTTT TGTAGAGATG GGGTTTCGCC ATGTTGGCCA GGCTGGCCTC    5460


 


GAACTGCTGA GCTCAAGCAA TCCGCCCGCC TCGGCCTCAC AAAGTCCTAG AATTTTAGGC    5520


 


ATGAGCCTCC GGGTCCGGCC TGTGCTAATC CTTTCTGTCC TTGGTTCTTT ATTTCTCTTC    5580


 


TCTCTTTTTC TTAGTCCCTT TTGTTCTTTC CCTCTCCCGT TCAGTTGGCT GTCGTTTGAG    5640


 


CCTCCACCTT TTCACTCCCT CCTTTCCACC ACGATGCCGA GCCCTGCCTT GGATGGGGAC    5700


 


CATCAGCGAT GACCACAATG ACCTCTCCCT TACCAGGCAG CTCCAGGCAG TGTTCCTGCA    5760


 


CCGCCTTTCC CAGGGCTTGG GGGCTTTTTC TAGTGGGCTT TGAGCTGCTC AATCTGGCCT    5820


 


CTGCAGGGCC GGCTCCCAGC CCTTCCAACC TCCTCACAGC CCGACCTGGG ACCTAGCCAA    5880


 


TTCCCGGAGA GTCTCTGTCC CATCGTGACC CCCTCACAAC TCTCCCACTC ACCAAAGTCT    5940


 


GATGACTGTG CTAGGGGGTG CTTATATAGA GTACTGAGTG TTACAAAAGC AGAAGTCTGG    6000


 


ATGAGAACCA ATTTGTGATA TTAAGCAGGT GGGGTGGGGG TGGGGAGTGT ACCTAGGTTC    6060


 


ATTTTCCGCC CTGCTTTTCC CCTTTCCAGT GTGTGCACTT AACCAGTCCC TGGGCCCTGT    6120


 


TCCCCATCCC CCTCCAAGGC ATGGATTGGG TGGGCTTGTG TGTCTTGGGG CAGGTGGCCC    6180


 


























TCTAGGGGCA GGAGTCAGCA GGGCATTAAC AAAAATAATT ACCATCCCCA CCCCCGACAG    6480


 


TGAAGTGGCT CTTTCCAGTT CACAGAGCAC TCTCACACCT CCCCGCTCTC ATTCTGGCCC    6540


 


TTCAGCTGAC TCGGACAAGC CAAGGATCTT GGTCCCCATT TTATAAAGGA GAAAACTGAG    6600


 


GCCCACGTGT AACAGTGATT GGCCCCAAGT CATCCCGGGA GCCAGCAGAA GAGCTAGGAC    6660


 


AGGAACCTAT TGTTCTAACT TCATATTGAT GCTAGCTTTT GACTATCCCT GAAACCGAGA    6720


 


TTGGTAATCA GCCCGGCTCT GAAACTGGTT ATTTGCTGGG GACTGTAAAA TAGGATTAAC    6780


 


TATTTCTAGT CCTGCATTTT AATTGCTGTT AGTAGGGCCA TCTTACCCAC CCTCTGAAGG    6840


 


ACCTGACTTG GCAAGCCCAA GGCAACATTC AGAATATGGC AGCTGAACCT CTGTGCACTT    6900


 


GTCTTTGGGC AGCAGCTGGG TCTTATTCTT CTCTGGCCTT CACAACATCC TGCAACCCAG    6960


 


CTCAAGGTCA GGAATGTGAC AGACTCATGT CATCATATCT CTGATGCCCA GAGAAGGGAT    7020


 


ACCATTTGCC TGAGCCTTCT CAGTACTGTT TAATCAGCCT GTGAGAACTT TCCTTGTGAA    7080


 


AGGCCCTGTC TGTGCCTGGG GCTGATAAAA CAGCAAGAAC GAACTGAGGA GCTGGGCAGC    7140


 


AGTGCAAAGC AAATACTACC AGCTTTGGTG CCTGTAAGTG TGGCTCTTAC TCATCTCACA    7200


 


TGGAAATAAG GGCAGCCACC TTGCAGGGCT GCTCTGAGGA TTGAGCTAAT ACAGTGCCCT    7260


 


GGGCGTTGGG GTGGGGAAAG TTGTGGAGCA CCTCCTGGGG GAAGGGGGTG TCAGAGCAGG    7320


 


GAATCTGGGG AGTCCGAGGG CACCTTCATC AACCCAATCT GTCATTTGAG CACCAGTCTT    7380


 


CACTGAGCCT CGTGGGCAAG CTGGAGGGAA ACAGGAATAA GGTCAGGCCC TGTTCTATAG    7440


 


GTCCCAGTGT AGTTGCTATG GTGAGTATCT TCATTTCCCT GCTTGCCCCA GCCACCTGGA    7500


 


GTGAGAAGCC CAAGAGGAAG CTGGGTGAGC TGTTTGTTTC CATGGGTCTC TGTGTTCACA    7560


 


GCTGACTCCC TTCACCAGCC AGCCCTTTCA CCTGAGCCCC AGCAACAAAG GCAGTCAGGC    7620


 


GGGGCTCAAA GCAGCTGCTC CAATGAAGTC AAAGAAATAA GCTCAGGGGA AGAAGCAGGT    7680


 


CACCCTCCCC CACTAGGGTG CTGGGCTCAC TTCCTCCTGG GGCAGTGGAG GAGGGTGTGG    7740


 


TTCCAACTCA GAACAAAATG GGGCTTTTGG TTTACTTTAT CACTCTTCAC AGCTCTGACC    7800


 


TGGACCCCTC ATCCCTGCCT GTCTTGTGGT GTAAGTGCGG ATCCCCCTAA GTTGGAGGAA    7860


 


AGGAAACTGG CCCAAACAAA AAGGAGAGCA GTTTTCTCTG CATCACATGG TAGGCCAGGA    7920


 


GGAGTCTAAT GCCCCAGAGT TTACTCTCAG CCCCCAAAAT CACCTAGCTA AATGTTACCT    7980


 


TATCTAAGAA GTCCTTAGGT TTTTTGGGGT TTGTTTTTTT TTTTTTTGAG ACAAGGTCTC    8040


 


ACTCTCTCAC CCAGACTGGA GCACAGTGGC ACAATCACAG CTCACTGCAG CCTCAACCTC    8100


 


CTGGGCTCAA GCAATCGTCC CAAGTAGCTG GGACTATAGG CCTGCACCAC CATGTCCAGC    8160


 


TAATTTATTT TTATTTATAT TTTTTAGACA GGGTCTCATT ATGTTGCCCT GGCTGGTCTT    8220


 


GAACTCCTGG GTTCAAGCAG TCCTCCCACC TCTGCCTCCC AAAGTGCTAG GTTTTTTTTT    8280


 


GTTTGTTTGT CTGTTTTTTG AAACAGAGTC TTGCTCTGTC GCCTAGGCTG GAGTGCAGTG    8340


 


GCACGATCTC AGCTACTGCA ACCTCCACCT CCTGGGTTCA AGTGATTCTC CTGCCTCAGC    8400


 


CTCCTAAGTA GTTGGGAATA CAGGCGTGTG CCAACACACC CAGCTCATTT TTGTATTTTT    8460


 


AGCGGAGATG GGGTTTTGCC ATGTTGGCCA AGCTGGTCTC AAACTCCTGA CCTCAGGTGA    8520


 


TTCGCCCGCC TCAGCCTCCC AAAGTGCTGG GTTTACAGGC GTGAGCCACC ACACCCAGCC    8580


 


CAAGAAGTCT TTTCTGATCA CCCACTCTTC CTTCTCTCCC AATGGCATTA GTTGTTCCCT    8640


 


CCTTTGCATT TTGAGAGTAT GTCCTGTAAG CCCCAAATGC AGCTTGAATC ATCTGCCCAT    8700


 


CCACCCCCTG TGCCCAACAG TAAGCCTCCT CTAGAGTAGA TACTATCTCC TGCATCTCAG    8760


 


TGAACCACTG CCCAGCAAAG CAGTCTTGCT AAAACAATGA CTCTAGAGAT CCTAAGCTGT    8820


 


GTGAGAGCTG GAGGAGAGAA TTAGACTGAT GGTCTGGGAA GGGATTGAAT TAGTCATCTT    8880


 


GTACCTTTTC TTCTTGACTT AAGTTCCAGA CCTGTAGCAA CCATTCCTGC TTAGACATCC    8940


 


AGAACATAAG CCTATGGGTC TGTGCCTGTT GGGTCTTAGT CTGGGTGAAA CTTTTCTCTA    9000


 


CTTCTGTCAG CTCTCCAGAT GAACCACAGA AGCAGGAATG TGGGCATCAT CAGTGAAATC    9060


 


TCTGCATACA GCAGACAAAG GGCTGGTCCA GTGGCTGTTT ATGAGGCAGC GCTAGGAGAG    9120


 


CTCTGATCCA GACTCTCCCT GCAGTGAAAG GGAGGGAGCC CTTCATGAAG TATTGACTGC    9180


 


TTGAGCAGGA ATTGCTTCAC CAGCACCTAA CTGAGTGCCT CTCGAGCTCA CATCGGTTTT    9240


 


CCCTCATGAG GCCACTTGGA GTCTTGCTGA GGGACTTGGT TCTATTAGGG AAGGTGAGTT    9300


 


TGGGGATGGT GAGCAGGGAG GGCCTGGGGA CATTGTGGCT AATGGGGCTT TTCTCCTCTT    9360


 


























ATGAAGCCTG GGAGTCTTGA TGGTGTCTAC TCTGTTCCCT CCCCAAAGAC ACAGACCCCT    9660


 


CAAGGGCCAG TGTTTGGAGC ATCGAGATGA CTGGAGGTGG GAAGGGCAAC ATGCTTATCC    9720


 


























TGAGTATGGA GCAAGGGTGT AGGCCCCTTG GCCCTTTTTT CTCAGTGATG ATTGATCTTA   10020


 


GTTCATTCAG CCATATAGTT TTTTAGGCCC CACGATCCCT AGGAAGATCA GGGGAACAGA   10080


 


GAACTGGAAG GGGCCCTGGT CCTCCACATA GTTCCTAAGC ACCTGGGCTA TACCAGGCTC   10140


 


TGAGCAGGGC GTCATCCCAT CACAGTCTTC AACACCACCT TGGGAGTAGG TAGTATCATC   10200


 


CCAGTGTTAT AGAAGAAGAG ACTGAGGTGG GAAGGCAGTG GGTAGAGTGG GGACTTGGCC   10260


 


AGGGGCACAC AGTAGAGAGC CAGAAAACAC ACAGTAGAGA GCCAGGACAC TCGTCTCTAA   10320


 


GGCCAGCGTT CTTCCCTTTC ACCTCCTTAG TATGCCATGC CAACCCTCCA TTTTACACAT   10380


 


GACGAAACAG AGCCCCAGAC AAAAGGTTGT CTTTCCCAGA TCACATGGCA GGAAGAAGTA   10440


 


AAGCTGACCT GAGATCCCAA GTCTTAGGAA TCCCAGTCCT CAGAAAGCCA CTTCTCTCTG   10500


 


AGCCTTGGTT TTCACATTTG TCAGATGGAA ATGATTGTGA TTTCTCAGGG CTGTTGAGCA   10560


 


GGTAAATGAA AATGTTTTAT GAAAGAAAGC ACCAAGTTTC ATTTTGGTCT TAGCCCTTGC   10620


 


TATGTCCCTA GCAAGAAGTA GATATTCATA GGGATATTTT GTTTGATGTG AGGAGTTCTT   10680


 


ACAGCAAGAG CTTGTAGAAG GCCAAAAGCT TCTGGATTCT ATTCCCAAAA GCAGGAGATG   10740


 


ACAGTGACAG GGTGGTTTTG GTGAGGAGAG ATGAGGTAGA AAATGAGTGC AAGCCCGCTG   10800


 


























CCCTTCCCCT TTTCCTCCTC CCCGATACTC TGGTCTCACC CAGGATCTTC CTCCTCCTAC   11100


 














ACGGTGCTGG GTCTCACCCT TCCCCCATGC CAGGAGCAGG TGCGGGGGTC TAGCTGACAC   11280


 


CAGAAGACCA CATCTTTTCA TCCTATTTGC CCTTTGCAGG GAGAGTAAGA TATCTCTTAC   11340


 


TTGCCATATT GAAGCCAATT GGGATGAAGC TCCCACTTTG CACATTGAGG AACTGAGGCT   11400


 


AGATTGGCAA AATGACTCTT TCAGGTCCTC AGAAGATGTC TCAGCTGGAG TCCCTGTCTG   11460


 


TTTTTGTTTT TTTGTTTGTT TGTTTTTTGT TTTTTTTGAG ATAGAGTCTC ACTCTGTTAC   11520


 


CCGTGTAATC TCAGCTCACT GCAACCTTCT CCTCCTGGGT TCAAGCGATT CTTGTGCCTC   11580


 


AGCCTCCCGA GTAGCTGGGA TGACAGGTGT GCACCAGCAC ACTGGCTAAT TTTTGTATTT   11640


 


TTAGTAGAGA TGGAGTTTCA CCATGTTAGC CAGGCTGGTC TCGAACTCCT GGCCTCAAGT   11700


 


GATCTGCCCA CCTTGGCCTC CCAATGTGCT GGGATTACAG GTGTGAGCCT CTGCGCCCCA   11760


 


TCCTCTTGTT TGTTTTTTGA GACAGGGTCT TGCTCGGTTG CCCAGGCTGG AGTGCAGTGG   11820


 


GGTGATTAAT GGCTCATTGC AGCCTCGACC TCCCTGACTC AAGCAATCCT CCCACCTCAG   11880


 


CCTCCTGAGT AGCTGGGGCT GACTACAGGC ATGCACACTG TGCCTGGCTA ATTTTTGTAT   11940


 


TTTGTAGAGA CAGGGTTTTT GCCATGTTAC CCAGTCTGGT CTTGAACTCC TGGGCTCAAG   12000


 


TGATCCACCC ACCTCGGCCT CCAAAAGAAG TCCTGGATTA CAGGCATGAG ACATTGTGCC   12060


 


CAGCCTCTCT GTCTCTTTAA AATCATGAAA ACTCGTAGCT ACTTAAGTAA TTCTCCTGCC   12120


 


























CTCTGGTCCT CCCCACCCAG TTCAGGCCTC TTGGCTATGC ATGTCTATTG TGGGTGGGAG   12420


 


AGAACCACCT GGAAGTGAGT AGCAGCCAAG TGTGACTATT TCTGATCCTG GTCCTGGCAT   12480


 


TTCACCAGCA TTCACCTATC CCCTTAATTC CTTCCTCCCA GAATTGCTAC CATCACTGTT   12540


 


TATTAGGTGT TAAATGGAGA CTCAAAGGGA ATTCATGCTT ATAGCCAAGC AGCTGTGAGC   12600


 


TCAGTTCATT GAGTCCTCCC AGCCTCCTTT GGGACAGAGC AACTGGGTTG GATTGAATAC   12660


 


CAGGCCCAGT GAGGGAAGTG GGAGGTGGAG GTGCCCCCAT GACCTGTGAT TTTTCTCCTC   12720


 


















































ACCAGAGGTC TTGGTCCAAT GGCCTTTTTG GTACCTGGTG GGCAGGGGAG GAACCACCTG   13260


 


ACTTTGAAAA TGGGTGTGAT CCACCTTCCA CCTCCAGCAT CCAATCTGAA GCCCGTGTAG   13320


 


GTCATCTGGT CCATTTCTCT CTAGACCCAG GCCCTGTACT AACATGGGGA GTGCAGGAGC   13380


 


CACCTGAGAG ACAGCAGTGC CTCCCCTTCC TTTGCCGGGC CACTTGAGCT CTTACTCAGA   13440


 


ATCTGGTACT CTAGTGCCTG CCATCCCAAC CCCCCACCCC AGCCGCAGGC CTGTTTATCT   13500


 


GCACAACAAG AGTGCTCCTG TGTGCCCTGC ATCTCCTGCA GTTCCAGAGG AACATGAGAC   13560


 


TCTTAGATGC TGTTGACTTT ATTTTATTCC ATTTTACAAA TGGAAGGAAG ACCCACCTCC   13620


 


CCCAAAGTCC CAGACCTTGT GAGAACAAGT CAGTCAGCCT CCTTCCACCC TCCACAGCCA   13680


 


CAGCCACACC CACAGAGGAA ATGTTACTGA ACTGGGTGGA GCAGGCCCTG ACTCCACAGA   13740


 


GGGTGGGTGG AGGCTGCAGG GCAAACATCT GGTCTCTGCC TGAGGATACT TTCCATTTGT   13800


 


GTTTTTTGTT GTTTTGAGAC AGAGTCTCAC TTGCTGTCAC CCAGGCTGGA GTGCAGTGGT   13860


 


GCAATCTTGG CTCACTGCAA CCTCTCCCAG GTTCAGGCGA TTCTCCTGCC TCAGCCTCCC   13920


 


AAGTAGCTGG GATTACAGGC ATACACCATC ATACCTGGCT AATTTTTGTG TTTTTGGTAG   13980


 


AAACGGGGTT TTGCCATGTT GGCCAGGCTG GTCTCAAACT CCTGACCTCA AGTGATCCAC   14040


 


CTACCTCAGC CTCCCAAAGT GCTGGGATTA CAGGCATGAG CCACTGTGCC TGGCCAGGAT   14100


 


ATTTTCCATT TGGAGTCTCA CCACCACAAC CCCCCTCCAC CTGCCCCTGC CCCAGCTAGG   14160


 


CATCCAAGGA GGCCGCAAGA AGCCAGGGCC TTGGCTGCAC AGGGGTCTCC GCTTCTCTGT   14220


 


CCCTGTTCTT ATCACCTGCA CTCAGAGGCA GGTGGGCAGG GGTACTACAA TTTCAAGGAG   14280


 


TGGAGACTGT GAGGTCCTGG AATCCCAAGG CATCTCCTGT AGGGCTGGGC CCTTAGAATT   14340


 


ATGTCACTCA GACCCAGTTT GTAGGTGTCT GAAGAAACTG AGGCCTGACA CAGGTGATGC   14400


 


AGGCAAGAAC ACCCAGAAAG TCCACTACTG AACTGGGACC GGGACCCAGT CCTCCTTCCC   14460


 


CTTGTGGACT CCCCCAGAGA CCAGTGCTGG GGTCCTTGGG GAAGCCTGTT TGGCAGCTGT   14520


 


GGAGCTAGGC CCTGAGAACA CGACCACCCT CCCTCTTCCC TCAGCCTCAA GCCGCTGAAG   14580


 


CCACTGCTGC TTCGCCGCCT CGTAAGCCCA ATGGTCAGAG CTGGAGGCTA GACCCTTCAG   14640


 


TGCTTGGGTT GAGGGCCAGG GTGTTAGATT GGTTTTTGGA GAAGGAACGA GGGCCCAGGA   14700


 


TTCTTCAGCT TCTTAGTTTT TGACAAATTG AGCTGAGGCC CCATAGTCCT CGGGAGGGAC   14760


 


AGGGTTGAGT GCCATAAGTC GGCAAACCAG GGTAAAGGTG ACAGGCAGCT CAGCCAGGCT   14820


 


GCAGGGGGTG GCATATACAG AGGACCTGGC CACTACTTTA TGTACCTTCT TACACTAATT   14880


 


CTGTGAGGCA GGCTGTTTGT TAGCTCTGCT CTGGACGGGA AGAAGTAGGG GCAGTTTGGT   14940


 


AGGTGTGTGT CAAAGCTAAA CAGGCTGGGT GGGCATGAGC AAGTCAGCTG GTTCATTCAG   15000


 


CAGCCTTAAT AGACACGAGG CTACCCAACT TCACTGTGGT TCTGGGTGTG GCCTTAGGAC   15060


 


AATGAGCTGG GAACAGTGGT AGGAACCACT GGAAAACATA CCAGTGGGTC TCATTCATTC   15120


 


TGATCACAGG TAGATCACTT CTCTTTGGTT CCCAACCCTT TAATGCCTAT TAAG         15174

## Claims

1. A method of modulating respiratory chain uncoupling in a cell or thermogenesis in a tissue, the method comprising contacting the cell or tissue with a miRNA agent that increases activity of at least one mitochondrial uncoupler selected from UCP1 or UCP2; wherein the miRNA agent is hsa-miR-515-3p or an agomir thereof.

2. The method of claim 1, wherein the cell is a pre-adipocyte, adipocyte, adipose tissue derived mesenchymal stem cell, hepatocyte, myocyte, or a precursor thereof.

3. The method of claim 1, wherein the tissue is brown fat, white fat, subcutaneous adipose tissue, liver or muscle.

4. The method of claim 3, wherein the tissue is contacted with the miRNA agent ex vivo.

5. The method of claim 1, wherein the mitochondrial uncoupler is UCP1 or UCP2.

6. The method of claim 1, wherein the miRNA agent is linked to targeting moiety.

7. The method of claim 6, wherein the targeting moiety is an aptamer.

8. The method of claim 6, wherein the targeting moiety delivers the miRNA agent to a specific cell type or tissue.

9. The method of claim 1, wherein the miRNA agent directly binds to the mRNA, 3′UTR, 5′UTR, coding, or promoter regions of UCP1 or UCP2.

10. The method of claim 1, wherein the miRNA agent modulates the activity of an activator or repressor of a mitochondrial uncoupling protein.

11. The method of claim 10, wherein the miRNA agent directly binds to the mRNA, 3′UTR, 5′UTR, coding, or promoter regions of the activator or repressor.

12. The method of claim 1, wherein the agent is an agomir of hsa-miR-515-3p.